Research Laboratory for Biotechnology and Biochemistry ( RLABB)

Calibration of Micropipettes

2011-08-17T06:42:00.001-07:00

Objective:

1. To know different parts of Micropipettes.

2. To learn how to use Micropipettes.

3. To calibrate micropipettes used in Molecular Biology laboratory.

4. To learn how to take care of Micropipettes.

Introduction:

Micropipettes used to accurately measure small volumes of liquids (volumes typically vary from 1 to 1000 µL). The parts of typical micropipettes are shown below.

Accuracy and Precision

Pipettes and micropipettes can deliver accurate and precise volumes of solution. Our goal is to determine how accurate and how precise our pipette and micropipette are.

Accuracy is a measure of how close a measured value is to the accepted or “true” value. It is related to the percent error between the average volume of solution measured experimentally and the volume that was expected (the accepted value). Smaller percent error reflects higher accuracy. Percent error can be negative, indicating that the measured volume was smaller than the expected volume or positive, indicating that the measured volume was larger than the expected volume. For example, we are attempting to measure two different volumes of water with our micropipette and two with our graduated pipette. Perfect accuracy would have us measure the exact volume we desire each time. However, the volume of water that we actually measure will be close but probably different from these volumes. The farther away from the correct volume, the lower the accuracy of our pipettes and/or our technique will be. The formula for percent error is in the Statistical Functions portion of the Lab Manual Introduction.

Precision measures the closeness of a set of values obtained from identical measurements of the same quantity. It is the ability to repetitively measure the same volume of solution (whether it’s accurate or not). Precision is related to the standard deviation of a series of measurements of the same thing. For example, if the micropipette is set to the same volume (300 µL) and four measurements are taken at this volume, a standard deviation can be taken of these five measurements. The smaller the standard deviation, the more precise the micropipette is. We will use the standard deviation as a measure of the spread of potential errors in a given measurement. The formula for standard deviation is in the Statistical Functions portion of the Lab Manual Introduction. Standard deviation is usually reported with the average value like this:

In order to minimize the waste generated from experiments in this class, a number of the experiments involve a micropipette that can deliver between 100 and 1000 microliters (µL). The micropipettes are only to be used for this volume range. For larger volumes, a graduated pipette is in your locker (bulbs available from the stockroom or one of the side drawers).

Use of the micropipettes

When you push down gently on the plunger of the micropipette, you will feel a “stop” where the resistance increases. If you push a little harder, the plunger will move even further to a second stop. The first stop is used to suck up the correct volume. The second stop is used to completely expel the liquid you are measuring.

Liquid is never drawn into the barrel of the micropipette itself. An appropriate tip should always be placed firmly on the end. Since the principle by which the micropipette works is the creation of a vacuum in the tip, causing liquid to be drawn up, it is critical that the tip be on tight enough to make an air-tight seal. Having said this, do NOT jam the tips on so hard that they are hard to get off. The tips used for the 1000µL pipettes are usually blue.

The volume to be taken up is set by turning the plunger on the top of the micropipette and reading the numerical settings displayed. A setting of 100 µL is equal to 0.100 mL. A setting of 1000 µL is equal to 1.000 mL. Do not set the micropipette below 100 µL or above 1000 µL under any circumstances! Doing this is essentially the only way that the micropipettes can be broken.

When drawing liquid up into the micropipette, set the dial, and then pushes the plunger down to the first stop. While holding it there, put the end of the tip under the surface of the liquid to be measured and slowly, gently allow the plunger to return to its top position. If you go too fast, you will cause some liquid to spurt up into the micropipette barrel itself, which is bad for the micropipette, bad for your results and bad for your chemistry karma, whatever that is. This also lets air in, so the volume of fluid sucked up into the tip will be lower than the amount that you want. Never let the plunger snap up by it. Next, place the end of the tip where you want the liquid to go and push the down the plunger to the second stop to deliver the exact amount of fluid desired.

Although micropipettes are usually quite accurate when first purchased, they can eventually develop problems with use. We will spend some time checking the calibration of the micropipettes that we will be using throughout the semester to ensure that they are delivering a known volume of fluid. We will measure out different exact volumes of water with the micropipette, and using the density of water at the temperature of the water, we will determine the volume of fluid delivered based on the fluids mass.

There are many techniques and tips available that will optimize your pipetting performance and increase the reproducibility of your results. A brief description of each follows:

The Equipment

1) Tips - It is advocated that only high quality tips which optimize the pipette’s performance be used. A high quality tip is one that has a smooth uniform interior with straight even sides that prevents the retention of liquids and minimizes surface wetting. Also, the tip should have a clean, hydrophobic surface and a perfectly centered opening in order to ensure the complete dispensing of the sample. These tips should always securely interface with the nosecone, because if they do not fit correctly, the amount of liquid dispensed can be dramatically influenced.

2) Liquid Viscosity - Since the pipette was originally factory calibrated using water, any liquid that has a viscosity higher or lower than water will impact the volume dispensed. Viscosity differentials should be accounted for and taken into consideration in order to enhance the accuracy of the instrument.

3) Container - The material of construction for the extraction vessel is also important, since some materials tend to force water into a convex configuration while other materials force water into a concave configuration. Obviously, this can impact the amount of liquid drawn into the tip. A glass container is recommended since it tends to force water into a concave configuration which helps to reduce or eliminate variations due to this effect.

The Operator

1) Technique - Most end users have a tendency to believe that the volume delivery is completely

dependent on the setting of the micrometer dial. Obviously, this is not the case, since many factors associated with pipettes come into play.

• Position - Pipettes should be held vertical during the aspiration of liquids,however, some end users often hold pipettes at many different angles during a pipetting interval. Holding a pipette 30o off vertical can cause as much as 0.7% more liquid to be aspirated due to the impact of hydrostatic pressure. Always store pipettes in an upright position when not in use.

• Pre-Wetting/Pre-Rinsing Tips -Failing to pre-wet tips can cause inconsistency between samples

since liquid in the initial samples adhere to the inside surfaces of the pipette tip, but liquid from later samples does not. Also, if a new volume is dialed in on the pipette’s micrometer, you will receive better results at the new volume by taking the old tip off and placing a new one on the shaft before you commence pipetting.

• Release of Plunger - Releasing the plunger abruptly can cause liquid to be “bumped”inside the pipette during a liquid transfer application. This can cause liquid to accumulate inside the instrument which in turn can be transferred to other samples causing variability in sample volume and the potential for cross contamination. It is recommended that a smooth, consistent pipetting rhythm be employed since it helps to increase both accuracy and precision. After the liquid has been aspirated into the tip, the pipette should be placed against the wall of the receiving vessel and the plunger slowly depressed. This will help all of the liquid in the tip to be dispensed. After a pause of about 1 second, depress the plunger to the bottom or blowout position (if equipped) and remove the pipette from the sidewall by utilizing either a sliding action up the wall or a brief movement away from the wall (called “touching off”).

• Immersion Depth - The pipette tip should only be inserted into the vessel containing the liquid of be transferred about 1-3mm. If the tip is immersed beyond this, the results could be erroneously high. This is due to the fact that liquid could adhere to the tip and be transferred along with the aliquot in the tip. If the tip is not immersed far enough then air could be drawn into the tip which could yield results that are incorrect on the low end.

• Equilibration Time – Troemner recommends that the tip, the pipette, the liquid being transferred, and the transfer container itself all be allowed to equilibrate to the same temperature. This is done to lessen the effects of thermal expansion which can dramatically impact the delivered volume.

• Thermal conductance – Thermal energy can be transferred from the operator’s hand to the air within the pipette (dead air) or even to the internal components themselves. This can have a dramatic impact on the amount of liquid dispensed due to the effects of expansion and/or contraction. To lessen this effect, it is recommended that some type of thermally insulated gloves like latex or cloth be worn.

2) Pipette Micrometer Setting – It is important to avoid significantly overdialing or underdialing the recommended range of the pipette. Volume delivery performance may change radically and may become completely undefined.

The Environment

1) Temperature – The volume delivery performance specifications of pipettes have been referenced by most manufacturers at room temperature which is defined as 20-25ºC. Any deviation from this specification can affect the amount of liquid dispensed due to the expansion or contraction of the internal components. Temperature is probably the most important factor that influences pipette performance. In fact, the density of water in a gravimetric analysis is calculated as a function of temperature.

2) Barometric Pressure – Pressure is reduced by 1.06" Hg for every 1000' of elevation, however, barometric pressure has only a small effect on the density formula, so the error encountered in not correcting for elevation is often ignored.

3) Relative Humidity – This is the percentage of moisture in the air at a measured dry bulb temperature compared to the amount of moisture that the air can hold at that temperature if the air is 100% saturated. Relative humidity exerts a major influence on taking accurate measurements of volume delivery. Under dry conditions, which are defined as less than 30% RH, it is extremely difficult to ensure an accurate measurement due to the rapid evaporation rate. Conversely, excessive humidity which is defined as greater than 75% can cause a measurement to be erroneously high due to condensation. Therefore, generally accepted guidelines for pipette volume delivery specify that relative humidity be maintained within the range of 45%-75%. Relative humidity also has an effect on the delivery of air displacement pipettes specifically. This is due to the evaporation of liquid from the upper several factors to consider when calibrating a pipette or choosing a calibration service:

1) If you require the “as found” data, it is advisable to obtain this before any parts or components are replaced since this can drastically change your results.

2) Clean and inspect the instrument for any visible signs of wear and tear. Make sure that the instrument can be autoclaved before autoclaving, since this can seriously damage the pipette.

3) Replace the pipette’s seals and o-rings and any other part that shows signs of wear. Remember

to pay special attention to the piston and replace if it seems especially worn or bent.

4) Ensure that the o-rings and seals have seated properly by performing a leak test and a vacuum test.

5) Allow the pipette to stabilize in an environmentally controlled, vibration-free room for a 24 – hour period to eliminate the effects of thermal expansion.

6) Decide which calibration technique that you wish to employ (i.e. Addition, Addition-Tare, Subtraction, or Subtraction-Tare).

7) Prepare the balance by “exercising” it and modifying it to accept a liquid containing vessel. It is our recommendation to use a glass container, so that the liquid has a concave meniscus.

8) Since most manufacturers originally calibrate their pipettes between 20-25°C while using bidistilled, degassed water, it is our recommendation that these conditions are duplicated.

9) Wear some type of thermally insulated gloves to lessen the transfer of heat from your hand to pipette. Latex or cloth seems to work the best.

10) Begin the liquid transfer stage of the calibration procedure utilizing the appropriate technique that you have chosen to employ.

11) Record the weightings, so that they can be converted into volumetric readings at the end of the calibration procedure.

12) Make the conversion taking into account all pertinent environmental conditions. Usually these conditions are used to calculate a Z-factor which is in turn used to convert from a mass reading to a volumetric reading.

Calibrating Micropipettes:

The two most common techniques of calibrating pipettes are the gravimetric and colorimetric (a.k.a. photometric) methods. Of these, the gravimetric method is the most common and the most widespread in use today. This method requires a stringently controlled environment, a high precision balance, a highly skilled pipetting technician, and a rudimentary understanding of statistics. The principle of this method is simple in that, given a certain mass of water with a known specific gravity; its volume can then be predicted. The accuracy and precision of the pipette can then be assessed by using an appropriate statistical approach. This method can be performed one of four ways: Addition, Addition-Tare, Subtraction, or Subtraction-Tare.

1) Addition is perhaps the most common mode of pipette calibration and it is performed by using the cumulative weight of a liquid to determine the volume dispensed.

2) The Addition-Tare method is performed by taring the balance each time before dispensing.

3) The Subtraction method uses the total subtracted weight of a liquid to determine the volume aspirated by the pipetting device. In this technique, you tare the balance only once, at the beginning, then you aspirate volumes of liquid from the vessel, take cumulative (negative) weights, and then calculate the volume aspirated based on the difference between the current and previous total weights.

4) The Subtraction-Tare method entails taring the balance each time before removing liquid from the vessel.

Since this method is not fool-proof, all variables must be stringently controlled and accounted for in order to produce results that are statistically accurate. The second most common type of pipette calibration process is the colorimetric or photometric method. This method involves the analysis of volumes of diluted dye in a cell of known path length. According to the Beer–Lambert Relationship, if a beam of monochromatic light passes through homogeneous solutions of equal pathlength, the absorbance measured is proportional to the dye concentration. So, with this in mind, an unknown volume of dye can be pipetted into a known volume of diluent, the resulting dye concentration can be measured photometrically, and the volume can be calculated. This method is less prone to environmental influences, but it requires the use of standardized consumables. Obviously, this means that each lot of standardized dye must be very carefully manufactured and calibrated in order to produce results of high accuracy. However, once solutions are prepared, calibrated and shown to be stable, accurate results can be obtained even at volumes less than one microliter11.

Principle:

The volume delivered by the pipettes is determined by weighing the amount delivered and dividing this by the density of water (at RT and 4°C).

Materials:

1. Balance (digital, upto 0.0001gm),

2. Micropipettes (1-10, 10-100, 100-1000µL),

3. Distilled water,

4. Aluminium foil

Procedure:

1. Turn on the balance

2. Place aluminium foil (prepared to cup shaped) on the pan of balance carefully

3. Zero the balance by tareing from its keyboard

4. Pipette out DW onto the foil and observe the weight of water on the data display

5. Zero the balance by tareing from its keyboard, again

6. Pipette out DW with the same pipette onto the foil and observe again the weight of water

7. Repeat steps 5 and 6 for more than 30 attempts

Observation:

Example: Pipetting of 10µL

Table: Weight (d=M/V) and corresponding volume (l) of water (d=1) delivered by micropipette

S. no.	1	2	3	4	5	6	7	…………………………………..	30
Experimental volume (µL)	11	12	09	10	13	09	08		11

Calculation/ Statistics:

Table 2: Determination of standard deviation (Sd) of volume of DW delivered by the micropipette

Experimental volume of DW (µL)-X	True Volume (µL) (expected volume)- X bar	(X-X bar)	(X-X bar)²
11
12
09
10
13
09
08
Calculate for all readings
11
S(X-X bar)²

Sd= Ö S(X-X bar)²/Ö n-1

Interpretation:

………………………………………………………………………………………………

Note: Ensure confidently for no handling error.

Reference:

1. Experiment 1: Volumetric Measurement; Using Micropipettes and Graduated Pipettes Adapted from the CSUS Biochemistry (Chem 162) Lab Manual, Fall 2002.

2. The Science Learning Center at the University of Michigan-Dearborn TROEMNER Pipette Sttandards handbook Raising the standard

3. Tiwari K.B and Ghimire P. (2010) A Practical Handbook for Microbial Genetics and Molecular Biology. First Edition, Kantipur College of Medical Sciences, Sitapaila, Kathmandu.

Biochemical Characterization of Yeast Isolates from Murcha

2009-08-08T07:16:00.000-07:00

Kiran Babu Tiwari1,2*, Manindra Lal Shrestha1, and Vishwanath Prasad Agrawal1,2

1Universal Science College, Pokhara University, Maitidevi, Kathmandu, Nepal
2Research Laboratory for Agricultural Biotechnology and Biochemistry, Maitidevi,
Kathmandu, Nepal

*Corresponding author: Kiran Babu Tiwari, Research Laboratory for Biotechnology and Biochemistry, Maitidevi, Kathmandu, Nepal. Email: kiranbabu.babukiran@gmail.com

Abstract

Eight Murcha samples were collected from different localities of Nepal. Altogether 31 yeasts (unicellular fungi) were isolated in Potato-Dextrose Agar (PDA). Compared to Saccharomyces cerevisiae (white/round/raised, Gluose/Fructose/Galactose/Sucrose/Maltose - positive, Mannose/Arabinose/Lactose/Citrate/Urea - negative), the yeasts were characterized morphologically and biochemically. Of the total isolates, eight (25.8%) were similar to S cerevisiae for their colonial properties. All isolates could assimilate Glucose, Maltose and Starch; and none of them could utilize citrate as carbon source. Higher proportions of the strains were able to assimilate Lactose (7, 22.6%), Mannose (6, 19.4%) and Arabinose (5, 16.2%) compared to S. cerevisiae. Further, nine (29.0%), five (16.2%) and one (3.2%) were unable to assimilate Galactose, Fructose and Sucrose respectively. Out of seven (22.6%) urea positive strains, two were able to hydrolyze urea strongly as nitrogen source.

2009-07-25T00:13:00.000-07:00

A Novel Class of Protease from Choerospondias axillaris (Lapsi) Leaves

Sudeep Karki1, Rupendra Shakya1 and Vishwanath P.Agrawal1,2

1Universal Science College and 2Research Laboratory for Biotechnology and Biochemistry (RLABB), Maitidevi, Kathmandu, Nepal.

Abstract

A novel protease from leaves of Choreospndias axillaris has been reported. C. axillaris , locally called Lapsi is dioceous, deciduous fruit – bearing large tree, having multiple daily uses. In an attempt to find method for determining sex of Lapsi at seedling stage , we stumbled upon a unique protease that has thwarted our effort to find sex - related protein. Thus protease is highly thermo – stable and acid resistant. Its preparation can be autoclaved without significant loss in activity. Its activity can be repeatedly precipitated by trichloroacetic acid. It possesses a Km value of 29 µM and Vmax 52.63 pmoles/min for bovine serum albumin as the substrate. It is catalytically so powerful that the level of soluble protein in leaf is below 20 µg per g dry weight. Lapsi leaf protease is an specific endopeptidase attacking peptide bonds that have phenylalanine, tyrosine, alanine and threonine / aspartic acid residues.

Introduction

Choerospondias axillaries:(Locally called “lapsi”), is a large, deciduous fruit-bearing tree of the family Anacardiaceae.A native of the Nepal hills (850–1900m) Lapsi wood is used as light construction timber and fuel wood; seed stones are used as fuel in brick kilns and the bark has medicinal value. Nepal is unique in processing and utilizing lapsi fruits. The fruits are rich in vitamin C content.

Agrawal and Kesari (1992) were first to observe strong proteotytic activity in Lapsi leaves. Lapsi Protease was found to be active even under autoclave condition. Dekhang and Sharma (2006) reported optimium pH of 7 for the protease. The protease is not inhibited at all by phenylmethanesulfonylfluoride ( PMSF ) and 20-30% inhibited by sodium iodoacetic acid, thus revealing that protease is not a serine protease. No smaller proteolytic products of BSA could be seen in SDS-PAGE using silver staining indicating that the protease is not exopeptidase, Protease activity can be repeatedly precipitated by 0.2 M tricloroacetic acid TCA (Singh and Giri 2007). In order to find an insight into the mechanism of protease action, the present research was carried out

______________________________

The research was done for the fulfilment of degree requirement for B. Sc. Biochemistry (SK and RS)

Materials and Methods

Preparation of Lapsi Leaf Powder : Dried leaves of lapsi were blended in a glass blender to get fine powder.

Partial purification and concentration of protease : It involved following steps.

1. Washing of the lapsi powder (5gm) with 50 ml acetone
2. Extraction of acetone washed powder with 10 ml phosphate buffer (0.1M of pH 7).
3. Heat treatment of the extract at 70. ºC for 30min
` 4. TCA precipitation of protein in the extract by adding TCA (2.45M) to heat treated solution to a final concentration of 0.2M followed by keeping in freeze for 10min, centrifuging at 10000rpm/10min and dissolving the pellet dissolve in 200μl of phosphate buffer.

Determination of proteolytic activity :

A typical reaction mixture cntaining 28.75μg of protein and 50 μg BSA in the total volume of 200μl of 0.1M pH 7 phosphate buffer was incubated for 30 min . The proteolytic activity is measured by 3 following methods.

1. Direct Method : The reaction was stopped by adding Bradford reagent for determination of BSA ( Bradford 1976, Saleemudin 1980 ).

2. Indirect Method : The reaction was stopped by adding 2.45M TCA to a final concentration is 0.2M, centrifuged at 10,000rpm/10min and pellet dissolved in 200μl of phosphate buffer (0.1M of pH-7) and protein determined by Bradford method.

3. Determination of amino acid produced in reaction .by Ninhydrin method :
To the reaction mixture (100μl), 0.9ml distilled water and 4ml Ninhydrin (0.5% in ethanol) reagent were added and heated at 80ºC for 10 min, cooled and absorbance measured at 570nm. Alanine was used as standard.

Detection of amino acids by paper chromatography :
Ascending paper chromatography of the reaction mixture (60μl 0.1% isopropanol) was done on Whatman paper 1 using Butanol: Glacial acetic acid: water (40:10:50 V/V) as the solvent system. Chromatogram was dried in air for 6 hrs.Amino acid spots were made visible by spraying the chromatogram with 0.2% Ninhydrin reagent. Then the purple coloured zones were marked and Rf value calculated.

Determined RF value of Standard Amino acid used.

Amino-Acids Rf-value Amino-Acids Rf-value
Alanine 0.35 Threonine 0.27
Tryptophan 0.63 Valine 0.8
Leucine 0.89 Aspartic acid 0.25
Iso-leucine 0.86 Serine 0.15
Phenylalanine 0.85 Cystine 0.05
Glycine 0.1 Arginine 0.07
Histidine 0.08 Glutamic acid 0.2
Tryrosine 0.45 Lysine 0.06

Results and Discussion

In order to measure the effect of enzyme concentration on protease activity, the double TCA precipitated enzyme (50μl) preparation (5.75ug/10μl) was used. It was noticed that enzyme activity was linear up to 28.75μg of protein. In order to determine the effect of time of incubation on protease activity, 50μl of double TCA precipitation enzyme (28.75μg) of protein and 50μg of the substrate (BSA) were used and incubated for various periods. Result obtained showed that the proteolytic activity was linear only up to 30min. Therefore, further experiments were carried out using 28.75μgm of protein and 30mins of incubation.In order to determine the Km and Vmax by Lineweaver-bulk plot, the effect of BSA concentration on enzyme activity was measured.. It was found that protease has Km of 29.1μM for BSA and Vmax of 52.63pmoles/min.A low value of Km indicates that the substrate is tightly bound to enzyme.

Since BSA has 607 amino acids , 607nmoles of amino acids should be produced per nmoles of BSA degraded. In order to check this equivalency, the amino aid contents of reaction mixture were analysed ( Table 1 ).

Table 1 : Comparison of experimental and the theoretical values of amino acid content of reaction mixture

BSA (μg)
a BSA degraded(μg)
b Theoretical value of amino acids produced
( nmoles)
c Experimental value of amino acids produced ( nmoles)
D c/d Calculated no. of free amino aacids
10 5.5 48.207 13.6 0282 171
20 12 105.180 28 0.266 161
50 26.8 234.904 65 0.277 168
100 60.4 529.410 145 0.273 166

*For BSA degradation, reaction mixture containing 28.75μg of enzyme and equired amount of BSA in total volume of 200μl of 0.1 M pH 7 phosphate buffer .After 30 min of incubation 100 μl of the mixture was used for protein determination using direct method.
*molecular weight of BSA 69323.4 Da, 1 mole of BSA contains 607 moles of amino acids
( Hilger et al. ) ; 50μg of BSA contains 0 .722 nmoles of BSA.
*For amino acid determination, to the reaction mixture (100 μl ) 0. 8.ml water was added and ninhydrin method used.

It was observed that upon proteolysis one molecule of BSA yielded only 161 -171 molecules of amino acids ( Table 1 ) suggesting that in addition to free amino acids 70 – 75 % BSA is degraded to smaller peptides which cannot be observed in SDS-PAGE as well as not amenable to the Ninhydrin method.

In order to determine the identity of released amino acids during proteolysis of BSA ,we embarked upon the paper chromatography of the reaction mixture . In paper chromatogram, only 4 spots could be seen. Comparing with the Rf-values of standard amino acids ( Fig. 1 ) we concluded that amino acids produced by degradation of BSA are phenylalanine, tyrosine, alanine and threonine / aspartic acid.

Table 2. Rf valuue of amino acids

Sample(enzyme) Rf-value Amino acid
Spot A 0.26 Thr , Asp
Spot B 0.35 Ala
Spot C 0.44 Tyr
Spot D 0.85 Phe

So, we conclude that lapsi protease is very special type of protease. It’s not a exopeptidase, it is an endopeptidase with very specific activity attacking peptide bonds containing phenylalanine, tyrosine, alanine, threonine / aspartic acid residues. A serious literature search shows that such type of protease has not been reported in the literature.

References

Agrawal VP, Keshari and Singh D, (1992) Study of Lapsi (Choreospondias axillaris) Protease Unpublished result.

Bradford MM (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry 72: 248-254.

Dekhang RN and Sharma G (2006) Study of Protease from the leaves of Choreospondias axillaries (Lapsi). Submitted to Universal Science College, Biochemistry Department for the fulfilment of degree requirement of . B. Sc. Biochemistry.

Hilger C, Grigioni F, De Beaufort C, Michel G, Freilinger J and Hentges F ( 2001 ) Differential binding of IgG and IgA antibodies to antigenic determinants of bovine serum albumin. J. Clin. Exp. Immupol. 123 (3), 387-394. http://www.ncbi.nlm.nih.gov/protein/3336842?report=genpep

Saleemudin M, Ahmad H and Hussain A. (1980) A Simple, Rpapid and Sensitive Procedure for the Assay of endoprotease using Coomassie Brilliant Blue G-250. Analytical Biochemistry 105: 202-205.

Singh R and Giri S (2007) Characterization and purification of protease from leaves Choerospondias Axillaris (Lapsi).Submitted to Universal Science College, Biochemistry Department for the fulfilment of degree requirement of B.Sc. Biochemistry.

Characterization of β-galactosidase from lactose utilizing yeast isolated from murcha

2009-07-17T01:19:00.000-07:00

Characterization of β-galactosidase from lactose utilizing yeast isolated from murcha

Shrestha KO¹, Adhikari S¹, Tiwari KB^1,2 and Agrawal VP^1,2

¹Universal Science College, Maitidevi, Kathmandu, Nepal

²Research Laboratory for Biotechnology and Biochemistry, Maitidevi, Kathmandu, Nepal

Abstract

Lactose utilizing yeasts have a potential application in the lactose pollution management in brewery and dairy industries as the organisms can grow at environmental conditions well. Eleven strains of lactose positive yeast were isolated from murcha samples collected from local markets in Kathmandu. Because of highest lactose positive activity among the isolated strains, L4 was selected for production, partially purification and characterization of β-galactosidase. Mass culture of the strain was treated with 2% (v/v) Chloroform to disrupt the cells. The enzyme was purified with Acetone precipitation method to 7.7 fold activity with 77.6% yield; specific activity was found to be 0.054 nmol/min/mg of protein. Optimum pH and temperature were determined to be 6.6 and 37°C respectively with the reaction time period 180 min. Michaelis-Menten constant (Km) value of the enzyme was found to be 8.798 mM of O-nitrophenyl-β-D-galactopyranoside.

Introduction

Murcha is a mixed starter inoculum, used in production of local alcoholic beverages in India, Tibet, Nepal, Bhutan etc. (Tsuyoshi et al., 2005). Murcha is a round cake, which is mildly acidic and has a pH around 5.2 containing 13% w/w moisture and 0.7% w/w ash (dry weight basis). The Murcha cakes contain mixed microbial population viz. molds, yeasts and bacteria (Tamang and Sarkar, 1995).

Yeasts are the world's premier industrial microorganisms, which have wide exploitation in the production of foods, beverages and pharmaceuticals. Alcoholic beverages are one of the major products in the world’s market. Yeasts can contaminate different dairy products because they have relatively low water activity (a_w, 0.88) (Frazier and Westhoff, 1995), can easily grow at room temperature and can utilize (assimilate and/or ferment) a variety of carbohydrates (Nahvi and Moeini, 2004), eg. pentoses, hexoses, disaccharides and, rarely polysaccharides (Barnett et al., 1990). The capacities of the organisms, thus, can be exploited to manage the biodegradeable wastes of the food, dairy and beverage industries. Most of the yeasts, except Saccharomyces spp., can grow on cellulosic materials, however; only few genera are able to degrade starch. Among disaccharides, lactose is one of the most refractory carbon substrate to most of the yeasts. Among hundreds of genera, only few are lactose positive, viz. many Cryptococcus spp. and Trichosporon spp.; some Debaryomyces spp., Kluyveromyces spp. and Myxozyma spp.; and occasionally Bullera spp., Candida spp., Rhodotorula spp. and Tremella spp. (Barnett et al., 1990). Lactose is one of the major components in whey in cheese industry that is non-friendly in the environment (Nahvi and Moeini, 2004). As they can grow at 25-30⁰C, yeasts can be exploited to manage lactose pollution (Sarova and Nikolova, 2002).

Yeast has been considered the predominant microbial enzyme source for food applications. Lactose utilizing yeast are good source of β-galactosidase. With rigorous search, Tiwari et al (2008) elucidated the presence of abundant lactose positive yeasts in murcha samples collected from various parts of Nepal. Hence, the study was designed to collect various yeasts capable to utilize lactose efficiently, and production, purification and characterization of β-galactosidase.

Materials and Methods

Isolation: Murcha samples were collected from local markets in Kathmandu, Nepal. The pure culture of yeast strains were isolated by serial dilution methods in Potato Dextrose Agar (PDA) (20% potato extract, 2% dextrose, and 1.5% agar, pH 4.5) incubating for 48 hr at 27⁰C. The purified isolates were stored on PDA slant at 4⁰C. The pure culture of the isolates was Gram stained for microscopic morphology.

Sugar assimilation test: The sugars used were glucose, fructose, sucrose, maltose, mannitol, galactose, lactose, and arabinose using a basal medium (4.5gm yeast extract, 7.5gm peptone and 20gm sugar in 1-lit distilled water) with phenol red (1mg/ml) as indicator. The pH was adjusted to 7.0-8.0. The medium was dispensed into tubes and sterilized by autoclaving. The pure culture of the isolates was incubated at 27 ⁰C till 72 hr. in the tubes and the result was indicated by change of color from red to yellow (Shrestha and Sharma, 1995). Control tubes were used in each set to monitor contamination.

Yeast Propagation: Sterile basal media containing ammonium sulphate, yeast extract and potassium dihydrogen phosphate was prepared with pH 4.5. Filter sterilized lactose (0.05M) was added to the 100 ml broth and yeast was inoculated and was incubated in waterbath shaker at 30°C for 48 hour.

Enzyme Extraction: The broth was centrifuged at 7000 rpm for 10 min. and the pellet was resuspended in 80ml phosphate buffer of pH 6.8. Chloroform (2%, v/v) was added and incubated overnight at 28°C. Incubated cells were centrifuged at 10000 rpm for 20 min. Supernatant was collected and one volume of acetone was added. The mixture was shaken in vertex shaker and centrifuged at 10000 rpm for 20 min. Pellets were collected and dissolved in 16ml of phosphate buffer with pH 6.8.

Enzyme Activity: β-galactosidase assay was done with O-nitrophenol β-D-galactopiranoside (ONPG) as substrate. Two test tubes were filled with 3ml of 0.01M of ONPG prepared in supplemented phosphate buffer of pH 6.8. Enzyme extract (1ml) was added to the test solution and reaction progress was determined spectrophotometrically against the blank tube at 420nm for every 30 min. for 3 hours. The reaction was quenched by adding 1ml of 0.5M sodium carbonate.

Determination of optimum time duration for reaction: Seven test tubes were filled with 3ml of 0.01M ONPG prepared in supplemented phosphate buffer of pH 6.8. Enzyme extract (1ml) was added in each tube and incubated at 37°C. By adding 1ml of 0.5M sodium carbonate, the reaction was quenched at every 30 min interval for respective tube and reaction progress was measured at 420nm against the blank.

Determination of optimum pH: Seven different test tubes were taken and 3ml of 0.01M ONPG prepared in buffer of pH 5.5, 6.0, 6.2, 6.4, 6.6, 6.8 and 7.0 was added to each respective test tube. Enzyme extract (1ml) was added to each test tube and incubated at 37°C for 180 min. The reaction was quenched by adding 1ml of 0.5M sodium carbonate and reaction progress was measured at 420nm against the blank.

Determination of optimum temperature: Seven test tubes were taken and 3ml of 0.01M ONPG prepared in phosphate buffer of pH 6.6 was added to each test tube. Enzyme extract 1ml was added to each test tube and incubated separately at 0°C, 15°C, 28°C, 37°C, 40°C, 45°C and 50°C respectively for 180 min. The reaction was quenched by adding 1ml of 0.5M sodium carbonate and reaction progress was measured at 420nm against the blank.

Determination of K_M value: ONPG solution (0.01M) was prepared in phosphate buffer of pH 6.6, and 0.6ml, 1.2ml, 1.8ml, 2.4ml, 3.0ml of the substrate was added in a series of five test tubes respectively. The final volume was set to 3ml by adding phosphate buffer of pH 6.6. Enzyme extract 1ml was added to each test tube and incubated at 37°C for 180 min. The reaction was quenched by adding 1ml of 0.5M sodium carbonate and reaction progress was measured at 420nm against the blank.

Results:

L4 strain those posses highest lactose positive activity was selected for production, partially purification and characterization of β-galactosidase. The enzyme was purified with Acetone precipitation method to 7.7 fold activity with 77.6% yield; specific activity was found to be 0.054 nmol/min/mg of protein (Table 1). Optimum pH and temperature were determined to be 6.6 (Fig. 1) and 37°C (Fig. 2) respectively with the reaction time period 180 min (Fig. 3). Michaelis-Menten constant (Km) value of the enzyme was found to be 8.798 mM of O-nitrophenyl-β-D-galactopyranoside (Fig. 4).

Discussion:

Yeasts are one of the important organisms having a wide industrial application. Most of the genera are unable to assimilate lactose as carbon source (Barnett et al., 1990). A higher abundance of the yeasts capable to produce β-galactosidase and thus assimilate lactose explored the importance of Nepalese yeast cakes (Murcha). Nahvi and Moeini (2004) reported three (3/30, 10.0%) β-galactosidase positive strains in Iran. In this study out of 148 isolates, only 11 were lactose positive.

The β-galactosidase from the yeast isolated from marcha on this study was characterized by their pH optimum at neutral or on the weekly acidic side. In general, β-galactosidase from yeast and bacteria have pH optimum near the neutral region, whereas the enzyme from molds act well at more acidic level as reported by Wierzbicki and Kosikowski (1973). The β-galactosidase of yeast was relatively heat labile, but they hydrolyze substrate well at lower temperature. The optimal hydrolysis was attended at 37°C and nearly 60% of hydrolyzing ability was maintained at 14°C. An enzyme workable at low temperature as possible is preferred for treatment of foods like milk. The Km value with ONPG as a substrate is slightly higher than reported in K. lactis by Kim and Lim (1981).

β-galactosidase commercially can be extracted from yeast. Its primary commercial use is to break down lactose in milk to make it suitable for people with lactose intolerance. β-galactosidase is also used in the manufacture of ice cream. Because glucose and galactose are sweeter than lactose, β-galactosidase produces a more pleasant taste. Lactose positive yeast strains can be used for removal of whey pollutants, Single cell protein (SCP) and ethanol production and treatment of lactose in dairy industry (Nahvi and Moeini, 2004).

Whey is the aqueous fraction of milk generated a by-product of cheese manufacturing which is produced in large amounts. The main solute in cheese whey is lactose present at a concentration of about 4.5-5% (Rohm et al., 1992; Zadow, 1992). Because of its high organic content, dumping directly to the environment causes serious contamination problems. As a solution, bioconversion of whey into SCP or ethanol has been performed in several countries (Gonzales, 1996; Irvine and Hill, 1985; Mawson, 1994). SCP could be produced from whey with employing of yeasts from different species including Kluyveromyces spp., Candida spp. and Trichosporon spp. as they are naturally able to metabolize lactose (Castillo, 1990). Although species of yeasts may differ considerably in their physiology, those of industrial importance have enough physiological characteristics in common to permit generalizations, provided that it is kept in mind that there will be exceptions to every statement made (Frazier and Westhoff, 1995). The findings explored that these yeasts may be new strains and lead to a search of yeast strains having high β-galactosidase activity (Nahvi and Moeini, 2004) as more efficient bioactive agents.

References

Barnett, J.A., D. Yarrow and R.W. Payne. 1990. The yeasts: Classification and identification. 2^nd Ed., Cambridge University Press. pp. 50-77.

Castillo, F. 1990. Lactose metabolism by yeast. In Verachtert, H. and R. DeMot (eds.) Yeast: Biotechnology and Biocatalysis. Marcel Dekker, New York; pp.: 297-320.

Frazier, W.C. and D.C. Westhoff. 1995. Food Microbiology. 4^th Ed., Tata McGraw-Hill Publishing Company Ltd. p10.

Gonzales, S.M.I. 1996. The biotechnological utilization of cheese whey. A review. Biores. Technol. 57: 1-11.

Irvine, D.M. and R.M. Hill. 1985. Cheese technology. In: Moo-young M (Ed.) Comprehensive Biotechnology. Pergman, Oxford; pp: 523-526.

Kim S.H. and Lim K.P. 1981. Differences in the hydrolysis of lactose and other substrates by β-D-Galactosidase from Kluyveromyces lactis.

Lodder, J. 1984.General classification of yeasts. In J Lodder (ed.), The yeasts. 3^rd ed., North-Holland Publishing Co., Amsterdam.

Mawson, A.J. 1994. Bioconversions for whey utilization and waste abatement. Biores. Technol. 47: 195-203.

Nahvi, I. and H. Moeini. 2004. Isolation and identification of yeast strains with high beta-galactosidase activity from diary products. Biotechnol. 3: 35-40.

Rohm, H., F. Eliskases-Lechner and M. Brauer. 1992. Diversity of yeasts in selected dairy products. J. Applied Bacteriol. 72: 370-376.

Sarova, I. and M. Nikolova. 2002. Isolation and taxonomic study of yeast strain from Bulgarian diary products. J. Culture Collection. 3: 59-65.

Shrestha, B. and A.P. Sharma. 1995. Manual on practical pharmaceutical microbiology. 1^st Ed., pp. 83-84.

Tamang, J.P. and P.K. Sarkar. 1995. Microflora of Murcha: an amylolytic fermentation starter. Microbiol. 81: 115-122.

Tiwari KB, Shrestha ML and Agrawal VPA. 2009. Abundance of lactose utilizing yeasts from Nepalese Murcha (Yeast Cakes). Nepalese Journal of Microbiology. In press

Tsuyoshi, N., R. Fudou, S. Yamanaka, M. Kozaki, N. Tamanag, S. Thapa and J. P. Tamang. 2005. Identification of yeast strains isolated from murcha in Sikkim, a microbial starter for amylolytic fermentation. Int. J. food Microbiol. 99: 135-146.

Wierzbicki, L. E., and F. V. Kosikowski. 1973. Food syrups from acid whey treated with/3-galactosidase of Aspergillus niger. J. Dairy Sci. 56:1182.

Zadow, J.G. 1992. Whey and lactose processing. Elservier Applied Science, London and New York.

Streptomyces coelicolor A3(2)

2009-07-16T07:26:00.000-07:00

Streptomyces coelicolor A3(2)

Streptomyces. These bacteria are widely distributed in nature, especially in the soil. The characteristic earthy smell of freshly plowed soil is actually attributed to the aromatic terpenoid geosmin produced by species of Streptomyces. There are currently 364 known species of this genus, many of which are the most important industrial producers of antibiotics and other secondary metabolites of antibacterial, antifungal, antiviral, and antitumor nature, as well as immunosuppressants, antihypercholesterolemics, etc. Streptomycetes are crucial in the soil environment because their diverse metabolism allows them to degrade the insoluble remains of other organisms, including recalcitrant compounds such as lignocelluloses and chitin. Streptomycetes produce both substrate and aerial mycelium. The latter shows characteristic modes of branching, and in the course of the streptomycete complex life cycle, these hyphae are partly transformed into chains of spores, which are often called conidia or arthrospores. An important feature in Streptomyces is the presence of type-I peptidoglycan in the cell walls that contains characteristic interpeptide glycine bridges. Another remarkable trait of streptomycetes is that they contain very large (~8 million base pairs which is about twice the size of most bacterial genomes) linear chromosomes with distinct telomeres. The linear chromosomal DNA is highly unstable and frequently undergoes large rearrangements at the extremities. These rearrangements consist of the deletion of several hundred kilobases, often associated with the amplification of an adjacent sequence, and lead to metabolic diversity within the Streptomyces group. Sequencing of several strains of Streptomyces is aimed partly on understanding the mechanisms involved in these diversification processes.

Streptomyces coelicolor. This bacterium is a soil-dwelling filamentous organism responsible for producing more than half of the known natural antibiotics. It is a well-studied species of Streptomyces and genetically is the best known representative.

Streptomyces coelicolor strain A3(2) M145. This strain is a derivative of the laboratory strain A3(2) lacking its two plasmids SCP1 and SCP2 which were sequenced separately after being isolated from the original strain A3(2).

SOURCE: http://www.blogger.com/post-create.g?blogID=8431478503600028158

Links:

1. http://www.blogger.com/post-create.g?blogID=8431478503600028158

2. http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=Retrieve&dopt=Overview&list_uids=242

3. http://www.ncbi.nlm.nih.gov/nuccore/30407153?report=genbank

4. http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=238

5. http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=Retrieve&dopt=Overview&list_uids=13298

6. http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=238

7. http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=241

8. http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=16388

GENETIC NOMENCLATURE

2009-07-15T09:16:00.001-07:00

GENETIC NOMENCLATURE

Source: http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/mutations/nomenclature-v3.pdf

Strain collections. The ease of rapidly accumulating a large number of mutants requires careful bookkeeping to avoid confusing one mutant with another. Each mutant should be assigned a strain number. Strain numbers usually consist of 2-3 capital letters designating their source and a serial numbering of the strains in a central laboratory collection. It is a good idea to check existing genetic resources to avoid the potential confusion that can result from assigning different genes the same name.

In addition to genome databases, good resources for gene names include the Salmonella Genetic Stock Centre (http://www.ucalgary.ca/~kesander/), the E. coli Genetic Stock Center (http://cgsc.biology.yale.edu/), and the Bacillus subtilis Genetics web site (http://www1.rhbnc.ac.uk/biological-sciences/cutting/index.html).

Nomenclature. Through the 1960’s, genetic nomenclature was a “tower of babel”. Due to the absence of clear rules for naming genes, each investigator assigned new names haphazardly, often resulting in the same name being applied to different genes or different names being applied to the same gene. To eliminate the resulting confusion, Demerec et al. (1966, 1986) developed a standard nomenclature for bacterial genes. With the development of new genetic tools, some modifications have been required. A detailed description of these rules can be found in the instructions to authors for the J. Bacteriol. (http://jb.asm.org/misc/ifora.shtml). The basic rules are described below.

GENOTYPE:

1. Genes. Each gene is assigned a three-letter designation, usually an abbreviation for the pathway or the phenotype of mutants. When the genotype is indicated, the three-letter designation is written in lower case. Different genes that affect the same pathway are distinguished by a capital letter following the three-letter designation.

For example, mutations affecting pyrimidine biosynthesis are designated pyr; the pyrC gene encodes the enzyme dihydroorotase and the pyrD gene encodes the enzyme dihydroorotate dehydrogenase.

2. Allele numbers. Each mutation in the pathway is consecutively assigned a unique allele number. A separate series of allele numbers is used for each three-letter locus designation. If there is no capital letter designating a specific gene, insert a dash before the allele number.

For example, pyrC19 refers to a particular pyr mutation that affects the pyrC gene. In order to distinguish each mutation, no other pyr mutation, regardless of the gene affected, will be assigned the allele number 19. A separate series of allele numbers is used for each three-letter locus designation. Allele numbers should be used sequentially and carefully monitored to insure that two different mutations are not named with the same allele numbers. The entire genotype is italicized or underlined (e.g. pyrC19).

3. Insertions. Transposable elements or suicide plasmids can insert in known genes or in a site on the chromosome where no gene is yet known. When an insertion is in a known gene, the mutation is given a three-letter designation, gene designation, and allele number as described above, followed by a double colon then the type of insertion element. DO NOT leave blank spaces between the letters or numbers and the colon. For example, a particular Tn10 insertion within the pyrC gene (mutant allele number 103) may be designated pyrC103::Tn10.

When a transposon insertion is not in a known gene, it is named according to the map position of the insertion on the chromosome. Such insertions are named with a three-letter symbol starting with z. The second and third letters indicate the approximate map position in minutes: the second letter corresponds to 10-minute intervals of the genetic map numbered clockwise from minute 0 (a = 0-9; b = 10-19; c = 20-29, etc.); the third letter corresponds to minutes within any 10-minute segment (a= 0; b = 1; c = 2; etc). For example, a Tn10 insertion located near pyrC at 23 minutes is designated zcd::Tn10.

Allele numbers are assigned sequentially to such insertions regardless of the letters appearing in the second and third positions, so if more refined mapping data suggests a new threeletter symbol, the allele number of the insertion mutation is retained. This nomenclature uses zaa (0 min) to zjj (99 min). Insertion mutations on extrachromosomal elements are designated with zz, followed by a letter denoting the element used. For example, zzf is used for insertion mutations on an F' plasmid. Insertions with an unknown location are designated zxx.

zaa = insertion at 0-1 min
zab = insertion at 1-2 min
zac = insertion at 2-3 min
zad = insertion at 3-4 min
zae = insertion at 4-5 min
zaf = insertion at 5-6 min
zag = insertion at 6-7 min
zah = insertion at 7-8 min
zai = insertion at 8-9 min
zaj = insertion at 9-10 min
zaa = insertion at 0 min
zba = insertion at 10 min
zca = insertion at 20 min
zda = insertion at 30 min
zea = insertion at 40 min
zfa = insertion at 50 min
zga = insertion at 60 min
zha = insertion at 70 min
zia = insertion at 80 min
zja = insertion at 90 min
zxx = insertion with unknown location
zzf = insertion on F-plasmid

Some commonly used mini-transposon derivatives are designated as follows:

Tn10dTet = Tet resistance, deleted for Tn10 transposase
Tn10dCam = Derived from Tn10dTet, Cam resistance substituted for Tet resistance
Tn10dKan = Derived from Tn10dTet, Kan resistance substituted for Tet resistance
Tn10dGen = Derived from Tn10dTet, Gen resistance substituted for Tet resistance
MudJ = Kan resistance, forms lac operon fusions, deleted for Mu transposase
MudJ-Cam = Derived from MudJ, Cam resistance marker disrupts Kan resistance
MudCam = Cam resistance substitution between ends of Mu

4. Plasmids. Plasmids should be indicated by a / slash after the genotype. Indicate the name of the plasmid, the plasmid origin, and the relevant genotype or phenotype carried by the plasmid. Insertions of suicide plasmids into the chromosome can be indicated as described for transposons. If a duplication is generated it can be described as indicated under chromosomal rearrangements.

5. Phage. Prophages or plasmids integrated into an attachment site can be indicated by the name of the attachment site followed by a double colon and the phage genotype indicated in brackets. For example, att::[P22 mnt::Kan].

6. Chromosome rearrangements. Chromosome rearrangements including deletions, duplications, and inversions should be indicated by a three letter symbol indicating the type of rearrangement, followed by the genes involved indicated in parenthesis, followed by the allele number.

Deletions = DEL(genes)allele number
Inversions = INV(join point gene #1 – join point gene #2)allele number
Duplications = DUP(gene #1*join point*gene #2)allele number

PHENOTYPE:

1. Growth phenotypes. It is often necessary to distinguish the phenotype of a strain from its genotype. The phenotype is usually indicated with the same three-letter designation as the genotype but phenotypes start with capital letters and are not underlined. (For example, strain TR251 [hisC527cysA1349 supD] has a Cys+ His+ phenotype because the supD mutation suppresses the amber mutations in both the cysA and the hisC genes.)

2. Antibiotic resistance. Both two and three letter designations are commonly used for antibiotic resistance markers. Both are acceptable, but it is essential to be consistent. Resistance and sensitivity is indicated with a superscript but on the computer it is often simpler to indicate resistance with (R) and sensitivity with (S).

Amp = Ampicillin
Cam = Chloramphenicol
Gen = Gentamicin
Kan = Kanamycin
Neo = Neomycin
Spc = Spectinomycin
Str = Streptomycin
Tet = Tetracycline
Zeo = Zeomycin
XG = X-gal
XP = X-phosphate

3. Conditional alleles. Conditional alleles indicated by the genotype including allele number followed by the two letter designation for the conditional phenotypes shown in parenthesis. For example, leuA414(Am). Note that because this is a phenotype it begins with a capital letter.

(Ts) = Temperature sensitive mutation
(Cs) = Cold sensitive mutation
(Am) = Amber mutation
(Op) = Opal mutation
(Oc) = Ochre mutation

REFERENCES:

Demerec, M., E. Adelberg, A. Clark , and P. Hartman. 1966. A proposal for a uniform nomenclature in bacterial genetics. Genetics 54(1):61-76.
Demerec, M., E. Adelberg, A. Clark , and P. Hartman. 1968. A proposal for a uniform nomenclature in bacterial genetics. J Gen Microbiol. 50(1):1-14.
Maloy, S., J. Cronan, and D. Friefelder. 1994. Microbial Genetics, Second edition. Jones and Bartlett, MA.
Maloy, S., V. Stewart, and R. Taylor. 1996. Genetic Analysis of Pathogenic Bacteria. Cold Spring Harbor Laboratory Press, NY.
Journal of Bacteriology Instructions to Authors. 2001. http://jb.asm.org/misc/ifora.shtml

Sodium Azide induced enhancement of antimicrobial property of previously selected actinomycetes (SIMA)

2009-06-10T22:27:00.000-07:00

A proposal on
Sodium Azide induced enhancement of antimicrobial property of previously selected actinomycetes (SIMA)

Principle Investigator: Pragya Sharma and Mandira Manandhar
Students from eighth semester
Universal Science College
Pokhara University
Maitidevi, Kathmandu 2009

Supervisor: Kiran Babu Tiwari
Universal Science College
Pokhara University
RLABBMaitidevi, Kathmandu

Research Lab
Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Maitidevi, Kathmandu

Introduction:
Actinomycetes, mostly known for their antibiotic activity, resemble both bacteria (prokaryotes) and fungi (eukaryotes). They have cell wall similar to gram positive bacteria with high G+C (>55%) content and also have 70S ribosome like that of bacteria. But their filamentous form, sporulating form and histone like protein (that are not found in prokaryotes) shows fungal characters. According to Bergey's Manual of Systematic Bacteriology, actinomycetes are divided into eight diverse families: Actinomycetaceae, Mycobacteriaceae, Actinoplanaceae, Frankiaceae, Dermatophilaceae, Nocardiaceae, Streptomycetaceae, Micromonosporaceae (Holt, 1989) and they comprise 63 genera (Nisbet and Fox, 1991). Characteristic biological aspects of actinomycetes is its ability to produce a wide variety of secondary metabolites including most of the antibiotics. Some examples of antibiotics produced actinomycetes are streptomycin, aureomycin, terramycin and chloromycetin.

Background:
In RLABB, Bhattarai and Tiwari (2006) developed a prototype methodology to explore mutagenic effects of sodium azide in Streptomyces spp., viz. both for loss of function (LOF) and gain of function (GOF) effects. The GOF is of particular importance and this type of work will be a good example of the foundation of applied research. The study should be extended in order to cover more parameters of actinomycetes, which helps to understand their physiology with respect to enhanced antibacterial properties among the mutants compared to respective wild types.

General objectives:
To study sodium azide induced enhancement of antimycrobial property of actionmycetes strain

Specific Objectives:
To revive and purify actinomycetes from preserved sample
To screen antibacterial activities of the strains by primary screening method
To verify the antibacterial activities of the strains by secondary screening method

Methodology Revival of the preserved actinomycetes: To revive them, they will be incubated at 28°C for one hour and then streaked on starch casein agar plates and further incubated at 28°C for 7 days.

Purification of actinomycetes : Streak plate method will be used to purify cultures of actinomycetes contaminated by bacteria and fungi (Williams and Cross, 1971, Singh and Agrawal 2002; Agrawal 2003). After isolation of the pure colonies based on their colonial morphology, colour of hyphae, color of aerial mycelium, they will be individually plated on another but the same agar medium.

Primary screening : Antibacterial activity of pure wild type and corresponding mutants will be determined by perpendicular streak method on Nutrient agar (NA). Test actinomycete strain will be streaked on the middle part of the NA plate and incubated for 5 - 7 days in room temparature. The test organisms to be used will be: Bacillus subtilis, Staphylococcus aureus, Enterobacter aerogens, Escherichia coli, Klebsiella species, Proteus species, Pseudomonas species, Salmonella typhi and Shigella species. Log phase culture (4hrs of NB at 37C) of the bacteria will be straeked perpendicular to the actinomycete streak line and incubated for overnight.

Secondary screening : Fresh and pure culture of each isolate selected from the primary screening will be inoculated in starch casein broth and incubated at 28°C for 7 days in water bath shaker . Growth of the organism in the flask will be confirmed by the visible pellets, clumps or aggregates and turbidity in the broth. Contents of flasks will be filtered through Whatman no.1 filter paper aseptically. The filtrate will be used for the determination of antibacterial activity against the standard test organisms by agar well-cut method.

Expected outcome: It has been found that azide induces gain of function and loss of function (Bhattarai and Tiwari, 2006). Enhanced antibacterial activity that they reported on primary screening (Bhattarai et al. 2007) is of particular importance; and hence, further elaboration of these applied aspects can be verified with secondary screening method. This study may explore some mutants with enhanced antibacterial properties compared to their respective wild types as a way of strain improvement among actinomycetes.

References:
Agrawal, V. P (2003) Biodiversity of Khumbu Region : Population Study of Actinomycetes, a Project Report Submitted to the Royal Nepal Academy of Science and Technology, Khumaltar, Lalitpur, Nepal "R"

Bhattarai, K., Tiwari, K.B. and Agrawal, V.P. (2007). Enhanced antibacterial activity of sodium azide treated mutant Streptomyces strain. Journal of Nepal Association for Medical Laboratory Sciences, 8(1): 67-8.

Bhattarai, K., Tiwari, K.B. and Agrawal, V.P. (2007). Loss-of-function (LOF) and gain-of-function (GOF) mutation of sodium azide in Streptomyces spp. Journal of Nepal Biotechnology Association. (accepted).

Holt, J.G., (1989) Bergey’s manual of systematic bacteriology, vol. 4, ed. S.T.Williams and M.E.Sharpe, Baltimore, Md : Williams and Williams.

Biochemical characterization of mutants generated by sodium azide treatment of corresponding sensitive wild Actinomycete strains isolated from Khumbu

2009-01-19T20:11:00.000-08:00

Biochemical characterization of mutants generated by sodium azide treatment of corresponding sensitive wild Actinomycete strains isolated from Khumbu, Everest Base Camp

Here goes below an abstract of work of Mamata Khatri and Yurika Rajbhandari who pursued their thesis conducted under my supervision and Prof. Agrawal as a partial fulfillment of their B. Sc. Biochemistry degree from Universal Science College, Pokhara University. They jointly worked to characterize biochemically the actinomycete mutants generated by sodium azide treatment of corresponding sensitive wild strains isolated from Khumbu, Everest Base Camp. The work is granted by International Foundation for Science (IFS) – 2008. This endeavor followed the findings of previous student, Keshav Bhattarai for his thesis work. With his work, we discovered various Gain-of-Function and Loss-of-Function mutations in a Streptomyces strain isolated from a soil sampele from Everest Base Camp.

Abstract: Sodium azide is being used to generate mutants of Actinomycetes isolated in Research Laboratory for Biotechnology and Biochemistry (RLABB) from soil samples from Khumbu, Mount Everest Base camp. This work was done to expand the limited number of actinomycete mutants sensitive 10-50ppm of sodium azide and characterize them biochemically alongwith the corresponding wild strains. Actinomycetes were isolated in Starch Casein Agar (SCA) from the soil samples and subsequently purified. Of the total 36 strains from RLABB, 24 were found to be sensitive to 50 – 100 ppm of sodium azide and were selected to generate mutants. Based on differences in colonial characteristics compared to the wild strains, 32 of mutants were selected and purified in an agar medium without the mutagen. The mutants and corresponding wild types were characterized biochemically and observed various Gain-of-Function (GOF) and Loss-of-function (LOF) mutation.

Generation of actinomycete mutants by exposing corresponding tolerant wild type strains from Khumbu, Everest Base Camp

2009-01-19T20:10:00.000-08:00

Here goes below an abstract of work of Junu Hamal and Shruti Regmi who pursued their thesis conducted under my supervision and Prof. Agrawal as a partial fulfillment of their B. Sc. Biochemistry degree from Universal Science College, Pokhara University. They jointly worked to generate mutants of tolerant wild type actinomycete strains isolated from Khumbu, Everest Base Camp. The work is granted by International Foundation for Science (IFS) – 2008. This endeavor followed the findings of previous student, Keshav Bhattarai for his thesis work. With his work, we discovered various Gain-of-Function and Loss-of-Function mutations in a Streptomyces strain isolated from a soil sampele from Everest Base Camp.

Abstract: Sodium azide is being used to generate mutants of Actinomycetes isolated in Research Laboratory for Biotechnology and Biochemistry (RLABB) from soil samples from Khumbu, Mount Everest Base camp. With the International Foundation for Science (IFS)-Grant 2008, this work was done to expand the limited number of mutants of tolerant actinomycetes (50-100ppm) of sodium azide. Actinomycetes were isolated in Starch Casein Agar (SCA) from the soil samples and subsequently purified. Of the total 36, 12 were found to be sensitive upto 100 ppm of sodium azide and were selected to generate mutants. Based on differences in colonial characteristics compared from the 12 wild strains, 36 mutants (each from 50ppm, 75ppm and 100ppm of sodium azide) were selected and purified in an agar medium without the mutagen. Individual mutants were described for their colonial morphologies.

Actinomycete Nomenclature system in RLABB

2009-01-19T20:09:00.000-08:00

Abstract: Sodium azide is being used to generate mutants of Actinomycetes isolated in Research Laboratory for Biotechnology and Biochemistry (RLABB) from soil samples from Khumbu, Mount Everest Base camp. With the International Foundation for Science (IFS)-Grant 2008, this work was done to expand the limited number of actinomycete mutants generated by sodium azide exposure. Feeling the need to develop the nomenclature system in order to designate and discriminate the vast array of the organisms’ database, we developed a “Guide for nomenclature of wild types and corresponding mutants of actinomycetes generated by exposure of sodium azide in RLABB” as follows:
1. Section 1: The wild strains are started with the capital initial of the region of isolation. E.g., S from Sagarmatha National Park, J from Jorsela, K from Kalapatthar and N from Namche.
2. Section 2: The capital letter for the wild type strain is followed by serial number which explains the different sites from the same region. E.g., S16 and S18 were isolated from two different sites of Sagarmatha National Park.
3. Section 3: The serial number may be tagged with decimal serial numbers to distinguish the soil samples from the same site from the same region. E.g., K70.1, K70.3 and K70.5 were the three isolates from corresponding three soil samples from the site (70th) of Kalapatthar region.
4. Section 4: The isolates from a soil samples are distinguished with the small letter tag after the serial numeral(s) described in sections 2 and 3 above. E.g., J1b and J1d are two isolates from the site (1st) from Jorsela region.
5. Section 5: Initials of the corresponding mutants are carried from the complete name of the respective wild types and followed by decimal and the concentration of sodium azide from which they were picked up. E.g., S18a.10 and S18a.20 were two mutants generated from S18a isolates exposed to 10ppm and 20ppm of sodium azide.
6. Section 6: Different mutants from the same concentration of sodium azide for the same wild type strain follows a decimal and i, ii, iii etc. E.g., S18a.10.i and S18a.10.ii were two mutant isolates from the S18a exposed to 10ppm of sodium azide.

Characterization of β-galactosidase from lactose utilizing yeast isolated from murcha

2009-01-19T20:07:00.000-08:00

Here goes below an abstract of Saket Adhikari and Krishna Om Shrestha who pursued their thesis conducted under my supervision (and Prof. Agrawal) as a partial fulfillment of their B. Sc. Biochemistry degree from Universal Science College, Pokhara University. They jointly worked to characterize β-galactosidase from lactose utilizing yeast isolated from murcha (Yeast Cakes) from Kathmandu valley. This endeavor followed the findings of previous student, Manindra Lal Shrestha for his thesis work. With his work we discovered the abundance of lactose utilizing yeasts in Nepalese murcha samples collected from different parts of Nepal; the paper is accepted in the second volume of Nepalese Journal of Microbiology (NJM).

Abstract
Lactose utilizing yeasts have a potential application in the lactose pollution management in brewery and dairy industries as the organisms can grow at environmental conditions well. Eleven strains of lactose positive yeast were isolated from murcha samples collected from local markets in Kathmandu. Because of highest lactose positive activity among the isolated strains, L4 was selected for production, partially purification and characterization of β-galactosidase. Mass culture of the strain was treated with 2% (v/v) Chloroform and the enzyme was purified with Acetone precipitation method to 7.7 fold activity. Specific activity of the enzyme was found to be 0.054 nmol/min/mg of protein, and Km value for O-nitrophenyl-β-D-galactopyranoside was 8.798 mM. Optimum temperature and pH were determined to be 37°C and 6.6 respectively with the reaction time period 180 min.

Study of Antibacterial Activity and DNA Polymorphisms of Streptomyces Mutants Developed By Sodium Azide Treatment.ppt

2007-11-03T22:37:00.001-07:00

A power point presented by my thesis students, Saroj and Looza, as their partial fulfillment of B. Sc. Biochemistry, Universal Science College, Pokhara University, 2007.

Supervisor: Kiran Babu Tiwari (gp120cdnashuffling@gmail.com)

Can be downloaded the ppt. from: http://www.mediafire.com/?4mojtlm9otf

Thermostable glucose isomerase from psychrotolerant Streptomyces species

2007-11-03T22:35:00.000-07:00

Bidur Dhungel1, Manoj Subedi1, Kiran Babu Tiwari1,2, Upendra Thapa Shrestha2, Subarna Pokhrel3 and Vishwanath Prasad Agrawal1, 2
1Universal science College, Pokhara University, Kathmandu, Nepal
2Research Laboratory for Agricultural Biotechnology and Biochemistry, Kathmandu, Nepal
3Department of Enzyme Engineering, Seoul National University, Korea
Corresponding Address: Dr. Vishwanath P. Agrawal, Professor of Biochemistry, Universal Science College, Pokhara University, Kathmandu. Email: vpa@wlink.com.np

Accepted in Journal of Nepal Biotechnology Association

ABSTRACT
Glucose isomerase (EC 5.3.1.5) was extracted from Streptomyces spp., isolated from Mt. Everest soil sample, and purified by ammonium sulfate fractionation and Sepharose-4B chromatography. A 7.1 fold increase in specific activity of the purified enzyme over crude was observed. Using glucose as substrate, the Michaelis constant (KM) and maximal velocity (Vmax) were found to be 0.45M and 0.18U/mg. respectively. The optimum substrate (glucose) concentration, optimum enzyme concentration, optimum pH, optimum temperature, and optimum reaction time were 0.6M, 62.14μg/100μl, 6.9, 70ºC, and 30 minutes, respectively. Optimum concentrations of Mg2+ and Co2+ were 10mM and 1mM, respectively. The enzyme was thermostable with half life 30 minutes at 100ºC.

Cloning of Bacillus thuringiensis cry3 fragment in Escherichia coli

2007-11-03T22:17:00.002-07:00

Shyam K. Shah1, Kiran Babu Tiwari11,2,3, Upendra Thapa Shrestha2, Subarna Pokhrel4 and Vishwanath Prasad Agrawal1,2*

1Department of Biochemistry, Universal Science College, Pokhara University, Kthmandu, Nepal,
2Research Laboratory for Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal,
3Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal,
4School of Chemical and Biological Engineering, Seoul National University, South Korea
*Corresponding author: Professor Dr. Vishwanath Prasad Agrawal, RLABB, E-mail: vpa@wlink.com.np, Tel: +977-1-2110043

Abstract
Bacillus thuringiensis was isolated and purified from the soil sample collected in Khumbu region of Mount Everest base camp. Total DNA was extracted, and PCR was done using nine universal
primers (Un1 to Un9) for cry1 to cry9 genes. A specific band of about 300 base pairs was amplified with universal primer Un3. DNA library was prepared into Escherichia coli HB101 using pUC18 vector and HindIII restriction site. Upon PCR screening of 1000 clones using Un3 primer, three clones possessed cry3 gene. The cry3 specific fragment was cloned, extracted and purified. As the bacterium was isolated from high altitude, the gene may have novel biological function.

Keywords: Bacillus thuringiensis, crystal protein, cry3 gene, screening

Introduction
Bacillus thuringiensis is an aerobic, ubiquitous, gram positive, spore forming bacterium that forms an insecticidal parasporal crystal protein (ä-endotoxin). The crystal protein, which is used to control insect pests of lepidoptera, diptera, coleoptera orders, is a useful alternative to synthetic chemical pesticide applied in commercial agriculture, forest management and mosquito control (Beegle and Yamamoto, 1992). The genes encoding ä-endotoxin production have been cloned in other bacteria and transferred into crop plants. This enables genetic improvement in the potency and host spectrum of B. thuringiensis strains and development of crop varieties that produce ä-endotoxin within their own tissues (Schnepf et al., 1998). The toxins are specific and have no detrimental effects on mammals or birds and are easily degraded in environment. In susceptible insects, the toxin is dissolved in the mid gut, releasing pro-toxin that are proteolytically converted into smaller toxin polypeptides (McGaughey and Whalen, 1992).
Following activation, these toxins bind with high affinity to receptors on the epithelium. After
binding, the toxins generate pores in the cell membrane, disturbing cellular osmotic balance and
causing the cell to swells and lyses. Recently, the crystal proteins and their genes have been
classified based on their structure, antigenic properties and activity spectrum.

Recently, the crystal proteins and their genes have been classified based on their structure, antigenic properties and activity spectrum. The proteins belonging to the Cry1 and Cry9 groups are toxic for lepidopteran insects. The Cry3, Cry7, and Cry8 proteins are active against coleopteran insects. The Cry5, Cry12, Cry13, and Cry14 proteins are nematocidal. The Cry11, Cry21, and Cyt proteins are toxic for dipteran insects. Currently, 45 different serotypes of B. thuringiensis have been classified as 58 serovars. Many Cry protein genes have been cloned, sequenced, and named cry and cyt genes. To date, over 100 cry gene sequences have been determined and classified in 22 groups and different subgroups with regard to their amino acid similarity. The proteins toxic for lepidopteran insects belong to the Cry1, Cry9, and Cry2 groups; toxins active against coleopteran insects are the Cry3, Cry7, and Cry8 proteins as well as the Cry1B and Cry1I proteins, which have dual activity. The Cry5, Cry12, Cry13, and Cry14 proteins are nematocidal, and the Cry2, Cry4, Cry10, Cry11, Cry16, Cry17, Cry19, and Cyt proteins are toxic for dipteran insects (Bravo et al., 1998).

In Nepalese context, though isolation and characterization of B. thuringiensis from different soil
samples and their insect toxicity have been studied and tested, molecular characterization of the
bacteria and the toxin has to be explored yet, especially from extreme environment in order to find novel strains. In an attempt to find novel crystal protein residing B. thuringiensis in high altitude, the bacteria were isolated from soil samples collected from Khumbu region of Mt. Everest base camp and processed in RLABB.

Materials and methods
Bacterial strains, plasmids and media: Bacillus thuringiensis of unknown strain was obtained from RLABB, Nepal. Plasmid pUC18 was used as cloning vector. Escherichia coli HB101 was used as host for cloning and expression of B. thuringiensis cry genes in Luria-Bertani medium (Bacto tryptone, 10g; yeast extract, 5g; NaCl, 10g per liter).

Isolation of total DNA: Genomic DNA from Bacillus thuringiensis was extracted from 50 ml
overnight culture (LB medium, 37°C, 150 rpm). Cells were harvested by centrifugation (8000 rpm, 5 minutes) and were resuspended in sterile SSC buffer. Lysozyme (10mg/ml) and Sodium dodecyl sulphate (10%) were used to lyse the cells and genomic DNA was extracted by phenol: chloroform method (Sambrook et al., 1989). DNA preparations were purified by Genei DNA purification kit (Genei, Banglore, India).

PCR using universal primers: PCR screening for cry genes in genomic DNA was performed by the method of Ben-Dov et al. (1997) using the universal primers (both direct and reverse each) Un1 (for cry1), Un2 (for cry2), Un3 (for cry3), Un4 (for cry4), Un5 (for cry5), Un6 (for cry6), Un7 (for cry7), Un8 (for cry8) and Un9 (for cry9) in a programmable thermal cycler (MJ Research, Inc.). For Screening Cry-group genes from the B. thuringiensis isolates PCR mixture was set up that contained PCR buffer (pH 8.3, 1X), dNTPs (100uM), forward primer (20pmol), reverse primer (20 pmol), Taqpolymerase (1U) and Genomic DNA (30-50 ng). Amplification was carried out in a DNA MiniCycler (MJ Research, Inc.,Watertown, Mass.) for 30 reaction cycles each as denaturation (1 min at 94°C) and annealing to primers (2 mins at 54 to 60°C), and at 72°C for 3 mins. The primers were obtained from University of British Columbia (UBC).
Genomic DNA library preparation: The pUC18 vector was digested by 2.5 µl of HindIII (5U/µl)
with 10µl of 10X buffer in 100 µl reaction mixture and was dephosphorylated with 5U of Calf intestinal alkaline phosphatase (CIAP). Similarly, 40 µl of B.thuringiensis genomic DNA was
digested with 5 µl of HindIII (5U/µl) with 10 µl of 10X buffer C in a 100 µl reaction mixture. The vector and inserts were purified using DNA purification kit and then ligated in 1:3 molar ratio. Vector, host strain, DNA purification and gel extraction kits, restriction enzyme, CIAP and other accessories were purchased from Genei (Genei, Bangalore, India) and the company’s instruction were followed accordingly in each step. Transformation was carried using chemically (0.1 M CaCl2) competent E. coli HB101 (Sambrrok et al., 1989), and the transformants were selected on LB-agar media containing ampicillin (100µg/ml). Each colony was streaked into LB-agar plate and the library of total 1000 representative colonies was formed.

Screening of cry3 genes: Each representative colony from the library was cultured in 5ml LB broth, and plasmid was extracted by alkaline extraction procedure (Sambrrok et al., 1989). DNA library was screened for cr3. The cry3 fragment (~300bp) was amplified by PCR and electrophoresed (2% agarose gel). The product was extracted, purified and stored in deep freeze.

Result
Among the nine universal primers used for PCR of the B. thuringiensis DNA, the cry3 gene fragment (~300) was found to be amplified using Un3 primers (Fig.1). In preliminary screening of 100 clones, cry3 were present in three colonies.

Fig-1. Agarose gel (2%) electrophoresis of PCR products amplified from total DNA from B.
thuringiensis strain with nine universal primers (Un). Lanes: C, negative control; M, Marker (ë
DNA/HindIII); 1, Un1; 2, Un2; 3, Un3; 4, Un4; 5, Un5; 6, Un6; 7, Un7; 8, Un8 and 9, Un9.

Discussion
The crystal proteins of B. thuringiensis have been extensively studied because of their pesticidal
properties. The increasingly rapid characterization of new crystal protein genes explored variety of sequences and activities of the crystal proteins. Isolation and characterization of B. thuringiensis from different soil sample and their insect toxicity have been studied and tested; molecular characterization of cry gene has not done yet in Nepal, especially from the B. thuringiensis strains collected from very high altitude. Hence, this study was planned with a hope to find novel B. thuringiensis that are cold tolerant, as it was isolated from Khumbu region (~4500m) of Mt. Everest base camp. The bacteria were found to possess cry3 gene in 100 of clones tested as a preliminary screening. The size of the cry3 fragment product (~300bp) is markedly lesser than reported elsewhere (589 to 604bp; Ben-Dov et al, 1997). The smaller size of the product may be due to loss of a part of the fragment during recombination events or may be novel cry3 gene fragment in the B. thuringiensis population in the Mt. Everest base camp. In a study, the crystal proteins extracted from some B. thuringiensis from the soil samples, where no mosquitoes are found, were found to be more mosquitocidal compared to that obtained from B. thuringiensis isolated from Kathmandu valley (Shrestha et al, 2006). The identification of known cry genes in the B. thuringiensis strains is important, since the specificity of action is known for many of the Cry toxins. This fact allows the possibility of selecting native strains that could be used in the control of some targets and of selecting strains with the highest activity. The information about the distribution of cry genes is limited. The characterizations done for most of the collections were based on bioassays against different insect larvae without identification of the cry genes present in the B. thuringiensis strains. In the last few years, some PCR-based methodologies have been proposed to identify different cry genes in B. thuringiensis strains. However, the cry gene list is increasing, and novel PCR primers are needed in order to identify some of the recently described cry genes (Bravo et al 1998).

More universal primers are to be tested against the B. thuringiensis DNA which may explore other cry genes. The absence of the PCR products doesn’t necessarily imply that the strain is devoid of respective gene. A strain may contain a novel gene not detectable with the universal primers for those bacteria, which are isolated from extreme ecological niches. A set of B. thuringiensis DNA library is being set in the RLABB which will help to explore putative
novel cry genes having broader entomocidal activities. The identification of putative novel B.
thuringiensis strains could be the first step in the sequence for finding novel toxicities, since novel toxins may be toxic for new targets. The isolation and sequencing of novel cry genes should be encouraged once the target insect is identified and more evidence on the potential of novel toxins as biological control agents is available (Bravo et al 1998).

References:
Beegle CC and Yamamoto. History of Bacillus thuringeinsis, Berliner research and development.
Can Entomol 1992; 124: 587-616.

Ben-Dov E, Zaritsky A, Dahan E, Barak Z, Sinai R, Manasherob R, Khamraev A, Troitskaya E,
Dubitsky A, Berezina N and Margalith Y. Extended screening by PCR for seven cry-group genes
from field-collected strains of B. thuringiensis. Appl Environ Microbiol 1997; 63: 4883-4890.

Birnboim HC and Dolly JA. Rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acid Res 1979; 7: 1513-22.

Bravo A, Sarabia S, Lopez L, Ontiveros H, Abarca C, Ortiz A et al. Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection. Appl Environ Microbiol 1998; 64(12): 4965-
72.

Johnson TM, Rishi AS, Nayak P and Sen S. Cloning of a cryIIIA endotoxin gene of B. thuringiensis var. tenebrionis and its transient expression in indica rice. J Biosci 1996; 21: 673-685.

McGaughey WH and Whalen ME. Managing insect resistance to Bacillus thuringeinsis Toxins.
Science 1992; 258: 1451-5.

Sambrook J, Fritsch EF and Maniatis T. Molecular cloning: A laboratory manual. 2nd Ed. Cold
Spring Harbor Laboratory press, Cold Spring Harbor, New York. 1989.

Schnepf HE and Whiteley HR. Cloning and expression of Bacillus thuringeinsis crystal protein gene in E. coli. Biochemistry 1981; 78: 2893-7.

Schnepf HE. et al. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev
1998; 62: 775-806.

Shreshtha UT, Sahukhal GS, Pokhrel S, Tiwari KB, Singh A and Agrawal VP. Delta- endotoxin
immuno cross-reactivity of Bacillus thuringiensis isolates collected from Khumbu base camp of
Mount Everest region. J Food Sci Technol Nepal 2006; 2: 128-131.

Comparative Study of Antibacterial Activity of Different Brands of Toothpastes Available in Nepal

2007-11-03T22:04:00.000-07:00

Principal Investigators: Amrit Acharya1, Bandana Subedi1, Basu Dev Paudyal1, Madav Jnawali1, Prabin Shakya1 and Upama K.C. 1
Supervisors: Upendra Thapa Shreshtha2, Kiran Babu Tiwari1, 2, Vishwanath Prasad Agrawal2*

1Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal
2Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Maitidevi, Kathmandu, Nepal
*Address for Correspondence: Prof. Dr. Vishwanath Prasad Agrawal, RLABB, Tel: +977-1-4442775, E-mail: vpa@wlink.com.np

Available as pdf: http://www.mediafire.com/?f2jmdq0x0zz

Toothpaste begins our days. Regular use of toothpaste maintains a good dental hygiene and health, which is its primary function. Toothpastes need to contain various antimicrobial agents in order to reduce, control and prevent different kinds of dental diseases. Different brands have their own composition and concentration of ingredients for their efficacy. These days, the markets are flushed with various attractive advertisements of their products from the corresponding manufacturers; however, the consumers should aware about the facts associated with their health. Hence, its an attempt to observe and assess the antimicrobial effects of different toothpastes available in Nepalese markets.

Objectives:

To isolate the organism responsible for dental plaque, dental caries and periodontal disease
To observe the antimicrobial effects of different brands of toothpaste (fluoridated and non- fluoridated) which are used in Nepal
To compare the antimicrobial effects of different tooth paste.

Chemoautotrophic bacteria on Methanogen isolation agar

2007-11-03T21:57:00.001-07:00

Strong mosquitocidal Bacillus thuringiensis from Mt. Everest

2007-11-03T21:37:00.000-07:00

Upendra Thapa Shreshtha1, Gyan Sunder Shahukhal1, Kiran Babu Tiwari1, Subarna Pokhrel3, Anjana Singh2, Vishwanath Prasad Agrawal1*

1Research Laboratory for Biotechnology and Biochemistry (RLABB), Maitidevi, Kathmandu, Nepal;
2Central Department of Microbiology, Tribhuvan University, Kirtipur, Nepal
3School of Biological Chemistry and Engineering, Seoul National University, S. Korea

*Address for Correspondence: Prof. Dr. Vishwanath Prasad Agrawal, RLABB, Tel: +977-1-4442775,
E-mail: vpa@wlink.com.np

ABSTRACT
Bacillus thuringiensis strains were isolated from soil samples collected from Khumbu Base Camp of the Everest region and characterized by standard microbiological techniques viz. colonial and morphological characteristics, and biochemical tests. Insect bioassay of each isolate was performed by standard method using mosquito larva. Among ten randomly selected isolates, one isolate showed the highest insecticidal activity against Dipteron insects.

Keywords: Insect-bioassay, Isolates, Khumbu region, Mosquitocidal, Mosquito larva

INTRODUCTION
Microbial insecticides are especially valuable as their toxicity to non-target animals and humans is extremely low compared to other commonly used chemical insecticides. They are safe for both the pesticide user and consumers of pesticide treated crops (Neppl, 2000). The soil bacterium, Bacillus thuringiensis, fulfills the requisites of a microbiological control agent against agricultural pest and vectors that cause massive crop destruction (Ben-Dov et al, 1999). The main target pest of B. thuringiensis insecticides include various Lepidoptera (butterfly), Diptera (flies and mosquitoes), and individual Coleopteran (Beatle) species and some strains kill off nematodes (Schnepf et al, 1998) where as B. thuringiensis var. kurstaki HD1 is highly potent strain due to its wide spread insecticidal properties (Dulmage, 1970).

METHODOLOGY
Soil sampling, isolation and biochemical characterization: Soil samples were collected from Sagarmatha National Park (SNP) and Phereche of Khumbu Base Camp of Everest region and were transported to RLABB, where the study was carried out from March 2005 to December 2005. B. thuringiensis were isolated by acetate selection method (Travers et al, 1987). The isolated organisms were identified by standard microbiological techniques including colonial and morphological characteristics, and biochemical tests (Bergey’s Manual, 1986).

Collection of Mosquito larvae: Mosquito (Lepidoptera) larvae were collected from the ditches in local area of Bode, Bhaktapur, Nepal for insect bioassay. The larvae were identified as Culex spp. by zoologists at the Central Department of Zoology, Tribhuvan University, Kirtipur and bioassay was performed as described by Pang (1994).

Insect bioassay: Larvae, collected from the ditches in local area of Bode, Bhaktapur, Nepal, were reared in a jar containing 100 ml of sterilized water containing 0.3 ml of 5% Brewer's Yeast and 5 ml of B. thuringiensis stationary phase culture and allowed to stand for 3 days. The number of deaths was recorded for one, two and three days. The crystal protein from the stationary culture of the selected individual strain was partially purified by Alkanine method (Dulmage, 1970) followed by Native-PAGE (Blackshear, 1984). The purified crystal protein was bio-assayed (30µg/ml per assay) from each band (Pang, 1994).

RESULTS
Out of 86 δ-endotoxin positive isolates, 10 randomly selected ones were used for insect bioassay. Although all isolates tested were effective against the larvae, isolate S6 was the most effective of all (Fig. 1).

DISCUSSION
Due to high toxicity of chemical pesticide to human beings, animals and beneficial insects, the use of chemical pesticides is being replaced by environment friendly bio-pesticides. The crystal proteins of B. thuringiensis, as bio-control agent, have been extensively studied worldwide. Under the present investigation, most of the bacterial strains were highly mosquitocidal. As the bacterial strains were collected from high altitude (above 4000m), where mosquitoes are not expected, the crystal proteins from the bacteria may be novel ones. In Nepalese context, though isolation and characterization of B. thuringiensis from different soil samples and their insect toxicity have been studied and tested from elsewhere, the characterization of crystal proteins for mosquitocidal properties hasn’t yet been explored from extreme environment. In an attempt to find novel crystal protein residing B. thuringiensis in high altitude, the bacteria were isolated from soil samples collected from Khumbu region of Mt. Everest base camp. The isolates showing potent insecticidal property tested against dipterans need to be studied further in larger trials so that it can have applicability to reduce the mosquitoes and different diseases caused by these vectors (Malaria, Filaria, Kala-azar etc).

ACKNOWLEDGEMENTWe express our full gratitude to CNR (Italy’s National Research Council) for supporting this work and especially thank to Mr. Deepak Singh, Mr. Yogan Khatri and Rajindra Aryal for collecting soil samples from Mount Everest region.

References:

Ben-Dov E, Wang Q, Zaritsky A, Manasherob R, Barak Z, Schneider B, Khamraev A, Baizhanov M, Glupov V, Margalith Y (1999). Multiplex PCR screening to detect cry9 genes in Bacillus thuringiensis strains. Appl Environ Microbiol; 65: 3714-6.

Blackshear PJ (1984). Systems for polyacrylamide gel electrophoresis. In Methods in enzymology (Jakoby WB eds.) vol 104: 237-255.

Claus D and Berkeley RCW (1986) ‘Genus bacillus. in: sneath PHA, ed. Bergey’s Manual of Systematic Bacteriology’, Baltimore: Williams & Wilkins, Volume. 2

Dulmage HT (1970). Production of spore-delta-endotoxin complex by variants of Bacillus thuringiensis in two fermentation media. J Invertebr Pathol; 16: 385-9.

Neppl CC (2000). Managing Resistance to Bacillus thuringiensis Toxins. Environmental Studies University of Chicago.

Pang AS (1994). Production of antibodies against Bacillus thuringiensis delta-endotoxin by injecting its plasmids. Biochem Biophys Res Commun ; 202: 1227-34.

Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR and Dean DH (1998). Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev; 62: 775-806.

Shrestha UT, Sahukhal GS, Pokhrel S, Tiwari KB, Singh A and Agrawal VP (2006). Delta- endotoxin immuno cross-reactivity of Bacillus thuringiensis isolates collected from Khumbu base camp of Mount Everest region. J Food Sci Technol Nepal; 2: 128-131.

Travers RS, Martin PA and Reichelderfer CF (1987). Selective Process for Efficient Isolation of Soil Bacillus spp. Appl Environ Microbiol; 53: 1263-6.

Biochemical and genetic Characterization of a Streptomyces sps

2007-07-27T04:02:00.000-07:00

A Thesis Proposal on

Biochemical and genetic Characterization of a Streptomyces sps from Everest region and chemical characterization of antibiotics produced therefrom

Principal investigator
Jyotish Yadav
Students from eighth semester
Universal Science College
Pokhara University
Maitidevi, Kathmandu
2007

Supervisor
Upendra Thapa Shrestha
Universal Science College
Pokhara University
RLABB
Maitidevi, Kathmandu

Research Lab

Research Laboratory for Biotechnology and Biochemistry (RLABB)

Maitidevi, Kathmandu

Introduction

Actinomycetes comprise an extensive and diverse group of Gram-positive, aerobic, mycelial bacteria with high G+C nucleotide content (>55%), and play an important ecological role in soil cycle. The name of the group actinomycetes is derived from the first described anaerobic species Actinomyces bovis that causes actinomycosis, the ‘ray-fungus disease’ of cattle. They were originally considered to be intermediate group between bacteria and fungi but are now recognized as prokaryotic microorganisms (Kuster 1968).
The majority of Actinomycetes are free living, saprophytic bacteria found widely distributed in soil, water and colonizing plants. Actinomycetes s population has been identified as one of the major group of soil population (Kuster 1968), which may vary with the soil type. They belong to the order Actinomycetales (Superkingdom: Bacteria, Phylum: Firmicutes, Class: Actinobacteria, Subclass: Actinobacteridae). According to Bergey's Manual Actinomycetes are divided into eight diverse families: Actinomycetaceae, Mycobacteriaceae, Actinoplanaceae, Frankiaceae, Dermatophilaceae, Nocardiaceae, Streptomycetaceae, Micromonosporaceae (Holt, 1989) and they comprise 63 genera (Nisbet and Fox, 1991). Based on 16s rRNA classification system they have recently been grouped in ten suborders: Actinomycineae, Corynebacterineae, Frankineae, Glycomycineae, Micrococineae, Micromonosporineae, Propionibacterineae, Pseudonocardineae, Streptomycineae and a large member of Streptomyces are still remained to be grouped (www.ncbi.nlm.nih.gov). Actinomycetes have characteristic biological aspects such as mycelial forms of growth that accumulates in sporulation and the ability to form a wide variety of secondary metabolites including most of the antibiotics. One of the major groups in actinomycetes is Streptomyces. It produces several antibiotics including of aminoglycosides, anthracyclins,glycopeptides, b-lactams, macrolides, nucleosides, peptides, polyenes, polyethers and tetracyclines (Sahin and Ugur, 2003).

The primary sources of these antibiotics are bacteria, Streptomyces, Nocardia and fungi with several classes of microorganisms contributing to the lesser extent. Thus investigators turn towards Streptomyces and also other genera of actinomycetes such as Nocardia, Micromonospora, Thermoactinomycetes etc. for isolation of novel antibiotics. No doubt soil is the natural habitat of most of the microorganisms where vast array of bacteria, actinomycetes, fungi and other organisms exist and provided with suitable growth condition and ability to proliferate. Thus most actinomycetes contributing to antibiotic production are screened from soil (Williams and Khan, 1974).

Our prime focus is to find out the novel antibiotic with broad-spectrum antimicrobial activity from Streptomyces isolates of high altitude. So in our research work we will proceed the Streptomyces isolates for further study from Khumbu region

Background

In, RLABB, The first work on the diversity of actinomycestes was started by Singh, D. and Agrawal, V.P. (2002). The research on actinomycetes form Mount Everest was then continued by Pandey, B., Ghimire, P. and Agrawal, V.P. (2004). Still the work is conducting by Rishi and Manita. Rishi found many actinomycetes with broad-spectrum antimicrobial activity. Among them most of actinomycetes are Streptomyces. Although more research have been done on antibiosis, classification of antibiotic groups and genetic characterization on the basis of 16s rDNA have to be done for significant use in medical field. Hence the study will explore more about the genetic property of Streptomyces and chemical property of antibiotics produced by them

Objectives

1. Biochemical and Genetic Characterization of Streptomyces
2. Chemical Characterization of antibiotics from the Streptomyces isolates

Methodology

1. Isolation Purification of Streptomyces
Soil samples will be obtained from Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB). Isolation of actinomycetes will be performed by soil dilution plate technique using Starch-Casein Agar (Singh and Agrawal, 2002 & 2003). Actinomycetes on the plates will be identified as colored, dried, rough, with irregular/regular margin; generally convex colony as described by Williams and Cross (1971). Streak plate method will be used to purify cultures of actinomycetes (Williams and Cross, 1971, Singh and Agrawal 2002; Agrawal 2003). After isolation of the pure colonies based on their colonial morphology, colour of hyphae, color of aerial mycelium, they will be individually plated on another but the same agar medium.

2. Morphological and Biochemical characterization: Morphological examination of the actinomycetes will be done by using cellophane tape and cover slip-buried methods (Williams and Cross, 1971; Singh and Agrawal 2002; Singh and Agrawal 2003). The mycelium structure, color and arrangement of conidiophores and arthrospore on the mycelium will be examined under oil immersion (1000X). The observed structure will be compared with Bergay’s manual of Determinative Bacteriology, Ninth edition (2000) for identification Streptomyces spp. Different biochemical tests will be performed to characterize the Streptomyces spp. The tests generally used are gelatin hydrolysis, starch hydrolysis, urea- hydrolysis, acid production from different sugars utilization tests, resistance to NaCl, temperature tolerance test, hydrogen sulphide production test, motility test, triple sugar iron (TSI) agar test, citrate utilization test, indole test, methyl red test, voges-proskauer (Acetoin Production) test, catalase test, oxidase test (Holt 1989; Singh and Agrawal 2002; Singh and Agrawal 2003).

3. Screening of Streptomyces for antimicrobial activity:

3.1 Primary screening

Primary screening of pure isolates will be determined by perpendicular streak method on Muller Hinton agar (MHA). In vitro screening of isolates for antagonism: MHA plates will be prepared and inoculated with Streptomyces isolate by a single streak of inoculum in the center of the petridish. After 4 days of incubation at 28 °C the plates were seeded with test organisms (Bacillus subtilis, Staphylococcus aureus, Enterobacter aerogens, Escherichia coli, Klebsiella species, Proteus species, Pseudomonas species, Salmonella typhi and Shigella species) by a single streak at a 90° angle to Streptomyces strains. The microbial interactions were analyzed by the determination of the size of the inhibition zone.

3.2 Secondary screening

Secondary screening is performed by agar well method against the standard test organism. Fresh and pure culture of each strain from the primary screening will be inoculated in starch casein broth and incubated at accordingly for 7 days in water bath shaker. Growth of the organism in the flask will be confirmed by the visible pellets, clumps or aggregates and turbidity in the broth. Contents of flasks will be filtered through Whatman no.1 filter paper. The filtrate will be used for the determination of antimicrobial activity against the standard test organisms.

4. Genetic Characterization

4.1 DNA extraction: Individual strains will be mass cultured in SC-broth by incubation the broth in shaker water bath for 5-6 days at 28C. Total DNA from corresponding strains will be extracted as described by a modified version of procedure of Kutchma et al. (1998) (Appendix-II).

4.2 DNA polymorphisms: DNA polymorphisms among the strains will be studied by Specific-PCR using Universal primers 8F and 1491R that will amplify specifically 16S rDNA. The amplified band will be sequenced for sub species identification (Rivas et al., 2001) (Appendix-III)

5. Fermentation process

Isolates showing the broad-spectrum antimicrobial activity are grown in submerged culture in 250 ml flasks containing 50 ml of SC Broth. The flasks are inoculated with 1ml of active Streptomyces culture and incubated at 28ºc for 120 hrs with shaking at 105 t/min.

6. Extraction of antimicrobial metabolites

Antibacterial compound is recovered from the filtrate by ethyl acetate extraction method following the process described by Westley et.al, 1979.and Liu et.al, 1986. Thus obtained compound is used to determine antimicrobial activity, minimum inhibitory concentration and to perform bioautography (Pandey et al., 2004).

7. Thin Layer Chromatography and Bioautography

Silica gel plates, 10 X 20 cm, 1mm thick, are prepared. They are activated at 150°C for half an hour. Ten microliters of the ethyl acetate fractions and reference antibiotics are applied on the plates and the chromatogram is developed using chloroform: methanol (4:1) as solvent system. The plates are run in duplicate; one set is used as the reference chromatogram and the other is used for bioautography. The spots in the chromatogram are visualized in the iodine vapour chamber and UV chamber Pandey et al., 2004).

Expected Outcomes

Being majority of antibiotics producing Actinomycetes are Streptomyces in this research work we have selected two Streptomyces sps with broad-spectrum antimicrobial activity. Since these species are from very cold Everest region the organisms as well as antibiotics produced by them may be novel ones.

References

Bergey's manual of determinative bacteriology 2000 Actinomycetales, 9th edition.

Holt JG 1989 Bergey's manual of systematic bacteriology, vol 4, ed. S.T. Williams and M.E. Sharpe, Baltimore, Md: Williams and Williams.

Kuster HJ, (1968) Uber die Bildung Von Huminstoffen durch Streptomyceten. Landwirtsch. Forsch 21:48-61

Kutchma AJ, Roberts MA, Knaebel DB and Crawford DL (1998) Small scale isolation of genomic DNA from Streptomyces mycelia or spores. Biotechniques 24: 452-457.

Nisbet LJ and Fox FM (1991) The importance of microbial biodiversity to biotechnology, In, The biodiversity of microorganisms and invertebrates: its role in sustainable Agriculture, ed.D.L. Hawksworth, 224-229, CAB International.

Pandey B, Ghimire P and Agrawal VP (2004) Studies on Antibacterial Activity of Soil from Khumbu Region of Mount Everest, a paper presented in International Conference on The Great Himalayas Climate, Health, Ecology, Management and Conservation, Kathmandu, January 12 -15

Rivas R., Velázquez E., Valverde A., Mateos PF and Molina EM (2001) A two primers random amplified polymorphic DNA procedure to obtain polymerase chain reaction fingerprints of bacterial species Electrophoresis 22: 1086–1089

Sahin N and Ugur A (2003) Investigation of the Antimicribial Activity of some Streptomyces isolates. Turk J Biol 27: 79-84.

Singh D and Agrawal VP (2002) Microbial Biodiversity of Mount Everest Region, a paper presented in International Seminar on Mountains - Kathmandu, March 6 – 8 (organized by Royal Nepal Academy of Science and Technology)

Singh D and Agrawal VP (2003) Diversity of Actinomycetes of Lobuche in Mount Everest I Proceedings of International Seminar on Mountains – Kathmandu: March 6 – 8, 2002 pp. 357 – 360.

Williams ST and Cross T (1971) Actinomycetes. In: J.R. Norris, D. W. Robbins, (eds), Methods in microbiology, vol.4. London, 295-334, Academic Perss, NewYork.

www.ncbi.nlm.nih.gov.

2007-07-10T21:47:00.000-07:00

A proposal on

Study of antibacterial properties of Streptomyces mutants developed by sodium azide exposure

Principle Investigator: Looza Shakya

Student from eighth semester

Universal Science College

Pokhara University

Maitidevi, Kathmandu2007

Supervisor: Kiran Babu Tiwari

Universal Science College

Pokhara University

RLABBMaitidevi, Kathmandu

Research Lab

Research Laboratory for AgriculturalBiotechnology and Biochemistry (RLABB)

Maitidevi, Kathmandu

Introduction:

Streptomycetes are complex multi-cellular saprophytic soil bacteria, perhaps best known for their ability to produce over two-thirds of naturally derived antibiotics currently in medical use (as well as many other valuable pharmaceuticals). They are gram positive organisms and assumed to be the transition group between fungi and bacteria (Agrawal, 2003). They belong to the order Actinomycetales (Super kingdom: Bacteria, Phylum: Firmicutes, Class: Actinobacteria, Subclass: Actinobacteridae). According to Bergey's Manual actinomycetes are divided into eight diverse families: Actinomycetaceae, Mycobacteriaceae, Actinoplanaceae, Frankiaceae, Dermatophilaceae, Nocardiaceae, Streptomycetaceae, Micromonosporaceae (Holt, 1989) and they comprise 63 genera (Nisbet and Fox, 1991). Based on 16s rRNA classification system they have recently been grouped in ten suborders: Actinomycineae, Corynebacterineae, Frankineae, Glycomycineae, Micrococineae, Micromonosporineae, Propionibacterineae, Pseudonocardineae, Streptomycineae and a large members of actinomycetes are still remained to be grouped (www.ncbi.nlm.nih.gov).

Actinomycetes have characteristic biological aspects such as mycelial forms of growth that accumulates in sporulation and the ability to form a wide variety of secondary metabolites including most of the antibiotics. Complex morphological development in the genera is phenotypic ally related to secondary metabolism (Hourinouchi and Beppu, 1992). Hence, investigation of actnomycetes from different ecological niches may yield novel isolates having more useful properties. Strain improvement for enhancement of antimicrobial activities holds a great significance in basic medical research.

Background:

In RLABB, Bhattarai and Tiwari (2006) developed a prototype methodology to explore mutagenic effects of sodium azide in Streptomyces spp., viz. both for loss of function (LOF) and gain of function (GOF) effects. The GOF is of particular importance and this type of work will be a good example of the foundation of applied research. The study should be extended in order to cover more parameters of actinomycetes, which helps to understand their physiology.

General objectives:

To develop mutants using sodium azide and study of comparative antibacterial activities among wild type and mutants

Specific Objectives:

To isolate and purify streptomycete from soil sample

To characterize the isolate for colonial, microscopical and biochemical properties

To develop mutants by treating the isolate with sodium azide in different concentrations

To characterize the mutants for colonial, microscopical and biochemical properties

To screen antibacterial activities of the strains by primary screening method

To verify the antibacterial activities of the strains by secondary screening method

Methodology:

Isolation of actinomycetes: Soil samples will be obtained from Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB). Isolation of actinomycetes will be performed by soil dilution plate technique using Starch-Casein Agar ( Singh and Agrawal, 2002 & 2003 ). Actinomycetes on the plates will be identified as colored, dried, rough, with irregular/regular margin; generally convex colony as described by Williams and Cross (1971).

Purification of actinomycetes: Streak plate method will be used to purify cultures of actinomycetes (Williams and Cross, 1971, Singh and Agrawal 2002; Agrawal 2003). After isolation of the pure colonies based on their colonial morphology, colour of hyphae, color of aerial mycelium, they will be individually plated on another but the same agar medium.

Morphological characterization: Morphological examination of the actinomycetes will be done by using cellophane tape and cover slip-buried methods (Williams and Cross, 1971; Singh and Agrawal 2002; Agrawal 2003). The mycelium structure, color and arrangement of conidiospore and arthrospore on the mycelium will be examined under oil immersion (1000X). The observed structure will be compared with Bergay’s manual of Determinative Bacteriology, Ninth edition (2000) for identification Streptomyces spp.

Biochemical characterization: Different biochemical tests will be performed to characterize the Streptomyces spp. The tests generally used are gelatin hydrolysis, starch hydrolysis, urea- hydrolysis, acid production from different sugars, resistance to NaCl, temperature tolerance test, hydrogen sulphide production test, motility test, triple sugar iron (TSI) agar test, citrate utilization test, indole test, methyl red test, voges-proskauer (Acetoin Production) test, catalase test, oxidase test (Holt 1989; Singh and Agrawal 2002; Agrawal 2003).

Exposure to sodium azide (Bhattarai et al. 2007): The isolate will be streaked on the Starch-Casein Agar plates containing varying concentrations (5-100ppm) of sodium azide, incubated at 28C for 5-6 days. Lethal concentration of sodium azide will be designated for that concentration that totally inhibits the growth. Mutants will be screened inintially based on differed colonial characteristics. Macroscopical, microscopical morphologies and biochemical characteristics of the corresponding mutants will be studied as described for wild strain.

Storage of wild and mutants strains: The mutants will be subcultured on azide free SCA (incubation at 28C for 5-6 days) plates and the strains will be stored in 15% glycerol containing Nutrient agar (G-NA) and keeping in deep freeze. The cultures will be revived as per the requirement.

Screening of Streptomyces strains for antimicrobial activity (Pandey et al. 2004):

a) Primary screening: The Streptomyces strains will be inoculated diagonally on the Nutrient agar (NA) plates and incubated at different temparatures, viz. 10oC, 20oC and 28oC. Antibacterial activity of the strains will be determined by streaking the test bacteria perpendicularly to the Streptomyces strains. The test organisms to be used will be: Bacillus subtilis, Staphylococcus aureus, Enterobacter aerogens, Escherichia coli, Klebsiella species, Proteus species, Pseudomonas species, Salmonella typhi and Shigella species.

b) Secondary screening: Fresh and pure culture of each strain from the primary screening will be inoculated in starch casein broth and incubated at accordingly for 7 days in water bath shaker. Growth of the organism in the flask will be confirmed by the visible pellets, clumps or aggregates and turbidity in the broth. Contents of flasks will be filtered through Whatman no.1 filter paper. The filtrate will be used for the determination of antibacterial activity against the standard test organisms.

Expected Outcome:

It has been found that azide induces gain of function and loss of function (Bhattarai and Tiwari, 2006). Enhanced antibacterial activity that they reported on primary screening (Bhattarai et al. 2007) is of particular importance; and hence, this applied aspect can be further elaborated and verified with secondary screening method. Possibly, the antibacterial activities can be created in those actinomycetes that do not posses the activity initially. Moreover, the mutants may develop different biochemical properties which will be of importance to undersatand more about streptomycetes’ physiology. Understanding such pathways and mutation of genes will give new idea for to further manipulate the organism.Moreover, this study can possibly reveal actinomycetes species that produce novel antibiotics. It is anticipated that efforts for the isolation, characterization and the study on actinomycetes can be a milestone for the discovery of antibiotics and novel species of actinomycetes.

References:

Agrawal, V. P (2003) Biodiversity of Khumbu Region : Population Study of Actinomycetes, a Project Report Submitted to the Royal Nepal Academy of Science and Technology, Khumaltar, Lalitpur, Nepal

Bhattarai, K., Tiwari, K.B. and Agrawal, V.P. (2007). Enhanced antibacterial activity of sodium azide treated mutant Streptomyces strain. Journal of Nepal Association for Medical Laboratory Sciences, 8(1): 67-8.

Bhattarai, K., Tiwari, K.B. and Agrawal, V.P. (2007). Loss-of-function (LOF) and gain-of-function (GOF) mutation of sodium azide in Streptomyces spp. Journal of Nepal Biotechnology Association. (accepted).

Holt, J.G., (1989) Bergey’s manual of systematic bacteriology, vol. 4, ed. S.T.Williams and M.E.Sharpe, Baltimore, Md : Williams and Williams

Pandey, B., Ghimire, P. and Agrawal, V.P. (2004 ) Studies on Antibacterial Activity of Soil from Khumbu Region of Mount Everest, a paper presented in International Conference on The Great Himalayas : Climate, Health, Ecology, Management and Conservation, Kathmandu, January 12 -15, 2004 ( organized by Kathmandu University and The Aquatic Ecosystem Health & Management Society, Canada )

Roberts MA and Crawford ( 2000) Use of Randomly Amplified Polymorphic DNA as a Means of Developing Genus- and Strain-Specific Streptomyces DNA Probes. Appl Environ Microbiol vol 66 2555–2564.

Singh, D. and Agrawal, V.P. (2002) Microbial Biodiversity of Mount Everest Region, a paper presented in International Seminar on Mountains - Kathmandu, March 6 – 8, 2002 ( organized by Royal Nepal Academy of Science and Technology )

Singh, D. and Agrawal, V.P. (2003) Diversity of Actinomycetes of Lobuche in Mount Everest I Proceedings of International Seminar on Mountains – Kathmandu, March 6 – 8, 2002 pp. 357 – 360.

2007-07-10T21:40:00.000-07:00

A proposal on
Study of DNA polymorphisms of Streptomyces mutants developed by sodium azide exposure

Principle Investigator: Saroj Sapkota
Student from eighth semester
Universal Science College
Pokhara University
Maitidevi, Kathmandu2007

Supervisor: Kiran Babu Tiwari
Universal Science College
Pokhara University
RLABB
Maitidevi, Kathmandu

Research Lab
Research Laboratory for AgriculturalBiotechnology and Biochemistry (RLABB)
Maitidevi, Kathmandu

Introduction:
Actiomycetes are the gram positive organisms and has been identified as one of the major groups of soil population. These organisms are assumed to be the transition group between fungi and bacteria. Actinomycetes are also called as antibiotic producing bacteria and such antibiotics are diverse in chemical structure. Actinomycetes belong to the order Actinomycetales ( Superkingdom: Bacteria, Phylum: Firmicutes, Class: Actinobacteria, Subclass: Actinobacteridae). According to Bergey's Manual of Systematic Bacteriology (1989), they are divided into eight diverse families: Actinomycetaceae, Microbacteriaceae, Actinoplanceae, Frankiaceae, Dermatophilaceae, Nocaveliaceae, Streptomycetaceae, Micromonosporaceae. Based on 16sr RNA classification system they have recently been grouped in ten suborders: Actinomycineae, Cornebacterineae, Frankieae, Micromonosporineae, Propionibacterineae, Pseudocardinieae, Streptomycineae.

Sodium azide is a mild mutagen and its toxicity is compared to cyanide. It has molecular formula NaN3 which consists of positive sodium ion and negative azide ion when mixed with water changes to gas. Thus it has very unstable configuration and tends to attain stable configuration of nitrogen gas. This property of molecule to attain stable configuration makes it to carry out different bio chemical manipulations which include the inhibition of enzymes, inhibition of oxidase, mutation, and also activate some biochemical pathways in living organisms.

Background:
In, RLABB, Bhattarai and Tiwari (2006) developed a prototype methodology to explore mutagenic effects of sodium azide in Streptomyces spp., viz. both for loss of function and gain of function effects. The study should be extended in order to cover more parameters of actinomycetes, which helps to understand their physiology.

General objectives:To develop mutants using sodium azide and study of DNA polymorphisms among wild type and mutants

Specific Objectives:
To isolate and purify streptomycete from soil sample
To characterize the isolate for colonial, microscopical and biochemical properties
To develop mutants by treating the isolate with sodium azide in different concentrations
To characterize the mutants for colonial, microscopical and biochemical properties
To extract total DNA from corresponding strains
To study the genetic polymorphisms among mutants and the wild type using
RAPD-primer 261 and 262 separately
RAPD primers 261 and 262 in combination

Methodology:
Isolation of actinomycetes: Soil samples will be obtained from Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB). Isolation of actinomycetes will be performed by soil dilution plate technique using Starch-Casein Agar ( Singh and Agrawal, 2002 & 2003 ). Actinomycetes on the plates will be identified as colored, dried, rough, with irregular/regular margin; generally convex colony as described by Williams and Cross (1971).

Purification of actinomycetes: Streak plate method will be used to purify cultures of actinomycetes (Williams and Cross, 1971, Singh and Agrawal 2002; Agrawal 2003). After isolation of the pure colonies based on their colonial morphology, colour of hyphae, color of aerial mycelium, they will be individually plated on another but the same agar medium.

Morphological characterization: Morphological examination of the actinomycetes will be done by using cellophane tape and cover slip-buried methods (Williams and Cross, 1971; Singh and Agrawal 2002; Agrawal 2003). The mycelium structure, color and arrangement of conidiospore and arthrospore on the mycelium will be examined under oil immersion (1000X). The observed structure will be compared with Bergay’s manual of Determinative Bacteriology, Ninth edition (2000) for identification Streptomyces spp.

Biochemical characterization: Different biochemical tests will be performed to characterize the Streptomyces spp. The tests generally used are gelatin hydrolysis, starch hydrolysis, urea- hydrolysis, acid production from different sugars, resistance to NaCl, temperature tolerance test, hydrogen sulphide production test, motility test, triple sugar iron (TSI) agar test, citrate utilization test, indole test, methyl red test, voges-proskauer (Acetoin Production) test, catalase test, oxidase test (Holt 1989; Singh and Agrawal 2002; Agrawal 2003).

Exposure to sodium azide (Bhattarai et al. 2007): The isolate will be streaked on the Starch-Casein Agar plates containing varying concentrations (5-100ppm) of sodium azide, incubated at 28C for 5-6 days. Lethal concentration of sodium azide will be designated for that concentration that totally inhibits the growth. Mutants will be screened inintially based on differed colonial characteristics. Macroscopical, microscopical morphologies and biochemical characteristics of the corresponding mutants will be studied as described for wild strain.

Storage of wild and mutants strains: The mutants will be subcultured on azide free SCA (incubation at 28C for 5-6 days) plates and the strains will be stored in 15% glycerol containing Nutrient agar (G-NA) and keeping in deep freeze. The cultures will be revived as per the requirement.

DNA extraction: Individual strains will be mass cultured in SC-broth by incubation the broth in shaker water bath for 5-6 days at 28C. Total DNA from corresponding strains will be extracted as described by Sambrook et al (1990).

DNA polymorphisms: DNA polymorphisms among the strains will be studied by RAPD-PCR using (a) Primer 261 (b) Primer 262 and (c) Combination of both primers (261+262). In the PCR mixture, 1U of Taq polymerase and 1% of DMSO will be used in addition to other regular components (1XPCR buffer containing 1.5mM MgCl2 with pH 8.3; dNTPs, 2.5mM each; 10-20ng of template DNA and primer/s, 10mM). The PCR products will be separated on a 1.5% agarose gel (0.5 µg/ml ethidium bromide) made in TAE buffer (pH 8.0).

Expected Outcome: It has been found that azide induces gain of function and loss of function (Bhattarai and Tiwari, 2006). The mutants may develop different biochemical properties which will be of importance to undersatand more about streptomycetes’ physiology. Understanding such pathways and mutation of genes will give new idea for to further manipulate the organism.

References:
Agrawal, V. P (2003) Biodiversity of Khumbu Region : Population Study of Actinomycetes, a Project Report Submitted to the Royal Nepal Academy of Science and Technology, Khumaltar, Lalitpur, Nepal

Bhattarai, K., Tiwari, K.B. and Agrawal, V.P. (2007). Enhanced antibacterial activity of sodium azide treated mutant Streptomyces strain. Journal of Nepal Association for Medical Laboratory Sciences, 8(1): 67-8.

Bhattarai, K., Tiwari, K.B. and Agrawal, V.P. (2007). Loss-of-function (LOF) and gain-of-function (GOF) mutation of sodium azide in Streptomyces spp. Journal of Nepal Biotechnology Association. (accepted).

Holt, J.G., (1989) Bergey’s manual of systematic bacteriology, vol. 4, ed. S.T.Williams and M.E.Sharpe, Baltimore, Md : Williams and Williams

Pandey, B., Ghimire, P. and Agrawal, V.P. (2004 ) Studies on Antibacterial Activity of Soil from Khumbu Region of Mount Everest, a paper presented in International Conference on The Great Himalayas : Climate, Health, Ecology, Management and Conservation, Kathmandu, January 12 -15, 2004 ( organized by Kathmandu University and The Aquatic Ecosystem Health & Management Society, Canada )

Roberts MA and Crawford ( 2000) Use of Randomly Amplified Polymorphic DNA as a Means of Developing Genus- and Strain-Specific Streptomyces DNA Probes. Appl Environ Microbiol vol 66 2555–2564.

Singh, D. and Agrawal, V.P. (2002) Microbial Biodiversity of Mount Everest Region, a paper presented in International Seminar on Mountains - Kathmandu, March 6 – 8, 2002 ( organized by Royal Nepal Academy of Science and Technology )

Singh, D. and Agrawal, V.P. (2003) Diversity of Actinomycetes of Lobuche in Mount Everest I Proceedings of International Seminar on Mountains – Kathmandu, March 6 – 8, 2002 pp. 357 – 360.

Proposed Research projects for 8th semester students

2007-06-13T21:26:00.000-07:00

1. Rupa Singh: Purification and characterization of protease from Lapsi. Supervisor: Vijayendra Agrawal

2. Mahendra Choudhary and Subha Giri: Commercial application of leaves of Choerospondias axillares as a source of heat stable protease. Supervisor: Rigzin Dekhang

3. Looza Shakya: Study of antibacterial properties of Streptomyces mutants developed by sodium azide exposure. Supervisor: Kiran Babu Tiwari

4. Saroj Sapkota: Study of DNA polymorphisms in Streptomyces mutants developed by sodium azide exposure. Supervisor: Kiran Babu Tiwari

5. Anil Chownhang: Screening for genetically modified food in Nepal. Supervisor: Prabhat Khadka

6. Deepmala Shrestha: Study of Cry genes of Bacillus thuringiensis strains by PCR. Supervisor: Gyan Sundar Sahukhala

7. Jyotish Yadav:

8. Sujan Gautam: Biochemical study of serum proteins from Buffalo (Bubalus bubalis) and its commercial utilization. [SDS-PAGE, Ammonium sulpahte, affinity chromatography (CNBr)]. Supervisor: Vijayendra Agrawal

2007-06-12T03:16:00.000-07:00

Delta-endotoxin Immuno Cross-reactivity of Bacillus thuringiensis Isolates Collected from Khumbu Base camp of Mount Everest Region

Upendra Thapa Shreshtha(1), Gyan Sundar Sahukhal(1), Subarna Pokhrel(1), Kiran Babu Tiwari(1), Anjana Singh(2), Vishwanath Prasad Agrawal(1*)
1Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Universal Science College, Maitidevi, Kathmandu, Nepal;
2Central Department of Microbiology, Tribhuvan University, Kirtipur, Nepal
*Address for Correspondence: Prof. Vishwanath Prasad Agrawal, RLABB, Tel: +977-1-4442775, E-mail: vpa@wlink.com.np

ABSTRACT

Bacillus thuringiensis strains were isolated from soil samples collected from Khumbu Base Camp of the Everest region and characterized by standard methods. Crystal protein (d-endotoxin) was extracted from the crystal protein producing strains (46 from Phereche and 40 from Sagarmatha National Park) from stationary phase culture broth and tested for insect bioassay. Crystal proteins were further purified by Native-PAGE. Among ten randomly selected isolates, one isolate showed the highest insecticidal activity against Dipteron insects. Its crystal protein had molecular weight about 120-130KD (revealed by SDS-PAGE) and was used to produce polyclonal antibody in New Zealand’s white rabbits. The presence of polyclonal antibody was confirmed by Ouchterlony double diffusion method. Indirect ELISA was optimized coating 6-8mg crystal protein per well in microtitre plate. The optimal dilution of the polyclonal antibody was 1000 folds corresponding to OD450 = 0.045 for color observation. Of the total 86 crystal protein producing isolates, crystal proteins from 31 isolates (36.05%) were 25-30% crossreactive, two groups of 6 isolates (6.97%) were 75-80% and 85-90% crossreactive respectively, and 4 isolates (4.65%) were 80-85% crossreactive with the polyclonal antisera. Only 3 isolates (3.49%) were more than 90% crossreactive. The Discriminatory Index (D) of the Indirect ELISA was 0.92.

Keywords: Khumbu region, d-endotoxin, Crystal protein, Insect-bioassay, Immunodiffusion, Immunocrossreactivity.

INTRODUCTION
Microbial insecticides are especially valuable as their toxicity to non target animals and humans is extremely low compared to other commonly used chemical insecticides. They are safer for both the pesticide user and consumers of pesticide treated crops (Neppl, 2000). The soil bacterium Bacillus thuringiensis fulfills the requisites of a microbiological control agent against agricultural pest and vectors that cause massive crop destruction (Ben-Dov et al, 1999). The main target pest of B. thuringiensis insecticides include various Lepidoptera (butterfly), Diptera (fliesand mosquitoes), and individual Coleopteran (Beatle) species and some strains kill off nematodes (Schnepf et al , 1998) where as B. thuringiensis var. kurstaki HD1 is highly potent strain due to its wide spread insecticidal properties (Dulmage, 1970).

Insect bioassay and rocket immunoelectrophoresis are currently used to detect and measure the levels of crystal proteins. Though the rocket immunoelectrophoresis is more sensitive than insect bioassay, it requires a considerable amount of antigen, at least 10 mg/ml (Wie et al, 1982). Immunodiffusion and ELISA are more practical and reliable methods. ELISA measures changes in enzyme activities proportional to the antigen or antibody concentrations. It is a highly versatile and sensitive analytical procedure for qualitative and quantitative determination of antibodies and almost any kind of antigens. The method discriminates different epitopes very efficiently provided that antibodies of high specificity and affinity are available. The detection limits of the assay may be well below 1 ng/ml (Perlmann and Perlmann, 2001). Hence ELISA can be effectively used to study cross reactivity of a given type of antigen. Identical antigens possess 100% crossreactivity with the given antisera and non identical ones don’t show any degree of crossreactivity. Thus the diversity of the given antigen and hence organisms in a given complex population can be studied by their cross reactivities. In order to study crystal protein diversity of B. thuringiensis strains, Indirect ELISA procedure was optimized in this study.

METHODOLOGY
Soil sampling, isolation and biochemical characterization: Soil samples were collected from Sagarmatha National Park (SNP) and Phereche of Khumbu Base Camp of Everest region and were transported to RLABB, where the study was carried out from March 2005 to December 2005 in joint collaboration with Central Department of Microbiology, Tribhuvan University, Kirtipur, Nepal. Bacillus thuringiensis were isolated by acetate selection method (Travers et al, 1987). The isolated organisms were identified by standard microbiological techniques including colonial and morphological characteristics, and biochemical tests (Bergey’s Manual, 1986).

Collection of Mosquito larvae: Mosquito (Lepidoptera) Larvae were collected from the ditches in local area of Bode, Bhaktapur, Nepal for insect bioassay. The larvae were identified as Culex spp. by zoologists at the Central Department of Zoology, Tribhuvan University, Kirtipur and bioassay was performed as described by Pang (1994).

Extraction and purification of crystal proteins: B. thuringiensis strains were incubated in Brain Heart Infusion broth (Calf brain infusion 200 g/l, beef heart infusion 250 g/l, protease peptone 10 g/l, dextrose 2 g/l, sodium chloride 5 g/l, disodium phosphate 2.5 g/l, final pH 7.4 ±0.2 at 25°C) and sterilized by autoclaving (15 lbs pressure 121oC, 15 min) at 30°C for 3-7 days till autolysis. Spores and crystals were separated by centrifugation (10,000 rpm, 20 min, 4 oC), and then washed four times with phosphate buffer (pH 7, 0.05M). The pellet was finally suspended overnight in carbonate buffer of pH 10.5 (0.05 M sodium carbonate, 0.01M b-mercaptoethanol, 1mM EDTA, 1mM PMSF) with constant shaking at 23-26oC, and centrifuged (10000 rpm, 20 min, 4 oC). Protein content in the supernatant was determined by Bradford assay (1976). In order to determine which proteins are responsible for the biological activities, they were electrophoresed under non denaturing conditions by Native PAGE (Blackshear, 1984) and the major bands were sliced , grinded in a minimum volume of phosphate-buffered saline (10 mM sodium phosphate, 150 mM NaCl, pH 7.2) , centrifuged (10000 rpm, 20 min, 4 oC) and concentrated using 20% TCA.

Insect bioassay and Molecular weight determination: For insect bioassay 10 larvae were taken in a jar containing 100 ml of sterilized water containing 0.3 ml of 5% Brewer's Yeast. Five ml of B. thuringiensis stationary phase culture was added and allowed to stand for 3 days. The number of deaths was recorded for one, two and three days. For the purified crystal protein bioassay, proteins (30µg/ml per assay) from each band were tested for insecticidal property as done by Pang (1994). Proteins from insect bioassay positive band was electrophoresed to determine molecular weight using molecular weight markers (lysozyme 14 KD, casein 22 KD, BSA 66KD) according to the
method of Laemmli (1970).

Polyclonal antiserum production: Proteins from insect bioassay positive band was resuspended in saline to a concentration of 1.0 mg/ml. The suspension was emulsified in an equal volume of Freund’s complete adjuvant (Difco, USA). A pair of New Zealand’s white rabbit was injected with 500 µg of the emulsified proteins (1.25 ml) by the intramuscular route in hind limbs. The booster injections with incomplete adjuvant were given three times in 14 days interval. The animals were bled 7 days after third booster dose. Polyclonal antiserum was pooled and decomplemented by incubation at 56°C for 30 min. Aliquots of antiserum (0.1 to 0.5 ml) were stored at -20°C until assayed.

Immunodiffusion and ELISA: The presence of polyclonal antibody was confirmed in a 1% agarose gel (0.05 M phosphate buffer, pH 7.2) by the Ouchterlony method (Talwar and Gupta, 1997) where 50 µl undiluted and diluted (1:10,1:100, 1:1000,1:10000 dilutions) antisera was poured in a centre well surrounded by 5 wells in petri-plate. 50 µl crystal protein antigen preparations (1 mg/ml) from SNP and Phereche isolates were applied in the surrounding wells against antiserum. The petri-plate was incubated at 4oC for 72 hours in a moist chamber. Immuno crossreactivity of B. thuriengensis crystal proteins was studied by indirect ELISA, where optimal dilutions of polyclonal antiserum and its second antibody (anti rabbit IgG conjugated with Horse Radish Peroxidase, Sigma, USA) was determined by chequerboard titration method (Trottier et al, 1972; Voller et al, 1976). To coat 96-well polystyrene microtitre plate, 100 µl crystal protein antigen (6µg) prepared in PBS (137 mM NaCl, 1.76 mM KH2PO4, 8.1 mM Na2HPO4, 2.7 mM KCl; pH 7.2 ) was applied in each well, and incubated overnight at 4oC (Trottier et al, 1972).

Uncoated protein was washed 3 times using washing buffer (0.05%Tween 20 in PBS). For blocking, 200µl of 2% BSA in PBS was applied in each well and allowed to stand for 1 hour at room temperature. Blocking was done twice. After 3 times washing, each well was incubated with 100 µl polyclonal antibody of different dilutions for 2 hours at room temperature, and unbound polyclonal antibody was washed 4 times. The microtitre plate was then incubated with 100 µl of second antibody of different dilutions per well at room temperature for 1 hour and washed 4 times. Finally it was incubated with 100 µl tetramethyl benzidine substrate solution (0.01% tetramethyl benzidine in citrate phosphate buffer of pH 4.9, 2µl of 30% H2O2), reaction was allowed proceed for half an hour, stopped adding 1N HCl and the intensity of color was read at 450 nm within 10 min in ELISA reader (Dynatech MR-250).

Calculation of discriminatory index value (D): D value was calculated according to the formula of Hunter and Gaston (1988).
Where, N=Numbers of isolates;
S=Number of different polymorphic types

RESULTS AND DISCUSSION

Strain identification
Total 109 B. thuringiensis isolates were obtained from soil samples of Phereche and SNP. Sixty three and 46 isolates were obtained from 52 Phereche and 39 SNP soil samples respectively. All the isolates were Gram positive rods. Of the 109 total isolates, only 86 isolates (79.63%) were positive on crystal protein staining, which were further proceeded for immunological characterization (Bergey’s Manual, 1986).

Molecular weight determination
Five bands were observed for S6 preparation having molecular weight of 40, 58, 71, 83 and 107 KD in SDS-PAGE and a single band (Mol. Wt. -120-130KD) was observed in Native PAGE. This indicates that S6 crystal protein is composed of 5 different protein subunits. Similar result was observed by Drobniewski and Ellar (1989) and Pfannenstiel et al (1986) in their work on B. thuringiensis.

Insect bioassay
The result of insect bioassay of crystal protein from SNP and Phereche B. thuriengensis isolates is displayed in Figure 1. For insect bioassay, cultures were grown to stationary phase which is suitable for the sporulation and production of crystal protein (Hunter and Gaston, 1988), and were used for the bioassay on mosquito larvae. Insecticidal activity of S6 endotoxin (30µg/ml) partially purified from autolysed B. thuringiensis broth by alkaline solution method following purification in Native PAGE is found to be 100 % (10/10) efficient, and hence it was used for polyclonal antibody production. Purification by Native PAGE has also been reported by Pang (1994). These crystal proteins are solubilized in the alkaline environment of Lepidoptera larval midgut, and then processing by midgut proteases results in a relatively stable, mature toxin (Van Rie et al., 1990). Activated cry toxins have two known functions, receptor binding and ion channel activity. The activated toxin binds readily to specific receptors on the apical brush border of the midgut microvilli of susceptible insects. Binding is a two-stage process involving
reversible and irreversible steps. The latter steps may involve a tight binding between the toxin and receptor, insertion of the toxin into the apical membrane, or both. It has been generally assumed that irreversible binding is exclusively associated with membrane insertion. Soon after insertion, toxins made pores on membrane and enter to body system. As soon as they enter to body they stop feeding and alternately death of insects occurs due to blood poisoning (Schnepf et al, 1998).

Immunodiffusion and ELISA
Double immunodiffusion performed in agarose gel gives clearly visible precipitin bands against crystal proteins of B. thuringiensis isolates from both SNP and Phereche upto 1:100 dilution of polyclonal antisera tested.(Fig. 2). The method gives reproducible results to detect crystal protein antigen with 100% specificity. For performing ELISA (Fig. 3), first and second antibody of dilutions 1:1000 and 1:2000 were optimal respectively. Below OD450 = 0.045, there was not clearly visible change in color to naked eyes. Trottier et al (1992) chose the optimal dilution of first and second antibodies difference between ELISA value with and without antigens with all other conditions remaining same. Between each ELISA step, plates were washed five times with a microtitre plate washer in order to prevent the false positive result (Trottier et al, 1992). The discriminatory index value, D (Table 1), was 0.92 (N=86, S=22). Hence the Indirect ELISA method divided all isolates into 13 groups with 92% confidence (Table-1). In which only 3 isolates (3.49%) were above 90% cross-reactive. The result clearly shows that B. thuringiensis isolates from Khumbu Base Camp are highly diverse for their crystal protein antigenicity. The S6 isolate showing potent insecticidal property tested against lepidopteron insects need to be studied further in larger trials so that it can have applicability to reduce the massive crop yield loss in this region.

CONCLUSION: B. thuringiensis is diversed into 13 different groups in Khumbu Base Camp of Mount Everest region. Crystal protein from one of the most potent strain (S6) is 100% effective against lepidopteron insects.

ACKNOWLEDGEMENT
We express our full gratitude to CNR (Italy’s National Research Council) for supporting this work and especially thank to Mr. Yogan Khatri, Mr. Deepak Singh and Rajendra Aryal for collecting soil samples from Mount Everest region. Finally my (Upendra Thapa Shrestha) special regards goes to my dear parents for their everlasting support.

REFERENCES
Neppl CC (2000). Managing Resistance to Bacillus thuringiensis Toxins. Environmental Studies University of Chicago.

Ben-Dov E, Wang Q, Zaritsky A, Manasherob R, Barak Z, Schneider B, Khamraev A, Baizhanov M, Glupov V, Margalith Y. Multiplex PCR screening to detect cry9 genes in Bacillus thuringiensis strains. Appl Environ Microbiol 1999; 65: 3714-6.

Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR and Dean DH. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 1998; 62: 775-806.

Dulmage HT Production of spore-delta-endotoxin complex by variants of Bacillus thuringiensis in two fermentation media. J Invertebr Pathol 1970; 16: 385-9.

Wie SI, Andrews R JR, Hammock B, Faust R.M and Bulla LA () Enzyme-Linked Immunosorbent Assays for Detection and Quantitation of the Entomocidal Parasporal Crystalline Protein of Bacillus thuringiensis subspp. kurstaki and israelensist Appl Environ Microbiol: 1982, 891-4.

Perlmann P and Perlmann H (2001) Enzyme-Linked Immunosorbent Assay ENCYCLOPEDIA OF LIFE SCIENCES 2001 Nature Publishing Group.

Travers RS, Martin PA and Reichelderfer CF Selective Process for Efficient Isolation of Soil Bacillus spp. Appl Environ Microbiol 1987; 53: 1263-6.

Bergey’s Manual of Systematic Bacteriology, Volume 2, 1986).

Pang AS. Production of antibodies against Bacillus thuringiensis delta-endotoxin by injecting its plasmids. Biochem Biophys Res Commun 1994; 202: 1227-34.

Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-54.

Blackshear PJ (1984). Systems for polyacrylamide gel electrophoresis. In Methods in enzymology (Jakoby WB eds.) vol 104: 237-255.

Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 1970; 227:680-5.

Talwar GP and Gupta SK (1997). A hand book of practical and clinical immunology, second edition, volume 1, CBS publishers and distributors, Daryaganj, New Delhi.

Trottier YL, Wright PF and Lariviere S Optimization and standardization of an Enzyme-Linked Immunosorbent Assay protocol for serodiagnosis of Actinobacillus pleuropneumoniae serotype 5. J Clin Microboil 1992;30(1):46-53.

Voller A., Bidwell D and Bartlett A. (1976) Microplate enzyme immunoassays for the immunodiagnosis of virus infections Manual of clinical immunology. American Society for Microbiology p. 506-512.

Hunter PR and Gaston MA. Numerical Index of the Discriminatory Ability of Typing Systems: an Application of Simpson's Index of Diversity. Journal of Clinical microbiology 1988; 26: 2465-6.

Drobniewski FA and Ellar DJ. Purification and properties of a 28-kilodalton hemolytic and mosquitocidal protein toxin of Bacillus thuringiensis subsp. darmstadiensis 73-E10-2. J Bacteriol 1989;171: 3060-7.

Pfannenstiel MA, Couche GA, Ross EJ and Nickerson KW. Immunological relationships among proteins making up the Bacillus thuringiensis subsp. israelensis crystalline toxin. Appl Environ Microbiol 1986;52: 644-9.

Van Rie, J., S. Jansens, H. Ho¨fte, D. Degheele, and H. Van Mellaert. Receptors on the brush border membrane of the insect midgut as determinants of the specificity of Bacillus thuringiensis delta-endotoxins. Appl. Environ. Microbiol 1990;56:1378-85.

Short Report

2007-06-12T03:05:00.000-07:00

LOF and GOF mutations in Streptomyces spp. by sodium azide

Keshav Bhattarai(1), Kiran Babu Tiwari(1, 2, 3*) and Vishwanath Prasad Agrawal(1)
1Department of Biochemistry, Universal Science College, Maitidevi, Kathmandu, Nepal
2Research Laboratory for Biotechnology and Biochemistry, Maitidevi, Kathmandu, Nepal
3Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal
*Address of correspondence: Kiran Babu Tiwari, Research Laboratory for Biotechnology and Biochemistry, Maitidevi, Kathmandu, Nepal, email:gp120cdnashuffling@gmail.com

Role of sodium azide as chemical mutagen has been studied in plants, fruit flies, rats etc. Several studies on loss of function (LOF) mutation and gain of function (GOF) mutation in different organisms could be seen, however GOF mutation is not yet reported in Streptomyces spp.Therefore, we attempted to explore on the beneficial mutations it can bring about while working with the actinomycetes isolates from Khumbu, Everest region.

A Streptomyces strain was isolated from a soil sample and purified at the Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB). The bacteria were then grown on plates containing 12.5, 25, 37.5 or 50ppm sodium azide. The sub-cultured colonies from 12.5ppm (S1), 25ppm (S2) and 37.5ppm (S3) containing plates were asporogenic and more yellowish, brittle, irregular and smaller in size compared to the wild strain (S0). Growth was not observed on plate containing 50ppm sodium azide indicating the lethal dose of sodium azide on the organism.

Treatment of Streptomyces with 12.5ppm of sodium azide enabled bacteria to utilize nitrate, whereas alternate carbon sources (sucrose, mannitol and salicin) were metabolized by those bacteria treated with 37.5ppm of the chemical. Thus the modified biochemistry of the treated Streptomyces strain could be due to the mutagenic effect of the Sodium azide.

To explore possible mutations in the treated Streptomyces strain, a RAPD primer (sequence CTGGCGTGAC; GC 70%; mp 34ºC) was used to investigate the DNA polymorphisms. Electrophoresis result in 1% agarose gel showed five bands in S0 while two bands were missing in mutant S3. The other mutants showed similar band patterns as of wild type. It was concluded that point mutations on the primer-binding site resulted in the loss of bands for S3. Furthermore, similar band patterns in other mutants with S0 may be due to point mutations between primer binding flanking ends, which went undetected. Hence, additional primer evaluation is required to identify potential differences in the S1 and S2 strains.

Further, studies on antibacterial activity of the mutants (viz. S1, S2 and S3) and wild strain (S0) were done against Staphylococcus aureus, Escherichia coli, Bacillus subtillis, B. thuringiensis, Pseudomonas aeruginosa, Klebsella pneumoniae and Proteus vulgaris. Marked increase in the antibacterial acivity as GOF mutation was observed against B. subtillis, a food poisoning bacterium.

In conclusion, these preliminary findings opened the possibilities of strain improvement with the use of chemical mutagen, which may have wider applications in the modern chemotherapy researches.

Enhanced Antibacterial Activity of Sodium Azide Treated Mutant Streptomyces Strain

2007-06-11T23:10:00.000-07:00

Enhanced Antibacterial Activity of Sodium Azide Treated Mutant Streptomyces Strain

Keshav Bhattarai(1), Kiran Babu Tiwari(1, 2, 3*) and Vishwanath Prasad Agrawal(1)
1Department of Biochemistry, Universal Science College, Maitidevi, Kathmandu, Nepal
2Research Laboratory for Biotechnology and Biochemistry, Maitidevi, Kathmandu, Nepal
3Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal
*Address of correspondence: Kiran Babu Tiwari, Research Laboratory for Biotechnology and Biochemistry, Maitidevi, Kathmandu, Nepal, email: babukiran@hotmail.com, Mob.: 9841374738

Abstract
A study was conducted to establish enhanced antibacterial activity, a Gain-of-Function (GOF) type of mutation, in Streptomyces strain exposed to sodium azide. The mutagenic concentrations of the azide were from 1.25–3.75mg%, beyond which the azide had lethal effect. The mutants (viz. S1, S2 and S3) and wild strain (S0) were screened for antibacterial activity against Staphylococcus aureus, Escherichia coli, Bacillus subtillis, B. thuringiensis, Pseudomonas aeruginosa, Klebsella pneumoniae and Proteus vulgaris. Marked increase in the antibacterial acivity was observed against B. subtillis. DNA polymorphisms were observed among the Streptomyces strains in a RAPD-PCR, which indicates mutagenic effect of sodium azide.

Keywords: Antibacterial activity, mutation, RAPD-PCR, sodium azide, Streptomyces spp.

Streptomycetes are aerobic or facultative anaerobic, gram positive, non-acid fast, non-motile with high “G+C” content (55%).1 Streptomyces spp. are the most populated and diverse soil bacteria producing various industrially and medically important secondary metabolites, such as enzymes (proteases, amylase, cellulase, lignase, chitinase etc.), chromogens and antibiotics. Streptomyces spp. produces most of the known antibiotics and, hence, is considered as the model research system that has therapeutic importance. The native isolate may not necessarily possesses potent antimicrobial activities; hence, strain improvement for enhanced useful activities holds a significant importance in basic medical researches. In various researches, sodium azide, a chemical mutagen, has been found to increase interleukin-2 production2 and guanylate cyclase activity3 in mammals. Here, Streptomyces strain was exposed to sodium azide to elucidate whether the natural antimicrobial activity of the strain is enhanced.

Streptomyces spp. was isolated from soil sample.4 The colored, dry, rough, with irregular/regular margin, convex colony was selected and subsequently purified. Starch casein agar (SCA) plates with 1.25mg%, 2.5mg%, 3.75mg%, 5.0mg%, 6.25mg%, 7.5mg%, 8.75mg% and 10.0mg% of sodium azide were prepared and 50µl of the pure culture was applied homogeneously on the surface of each plate. Control plate didn’t contain azide. The plates were incubated at 270C for 5 days. The corresponding azide resistant strains were sub-cultured onto sterile SCA plates without sodium azide. The test strains of the isolate were screened for antibacterial activities on nutrient agar plate by “Cross- streak plate” technique against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, B. thuringiensis, Pseudomonas aeruginosa, Klebsiella pneumoniae and Proteus vulgaris.

A corresponding reduction of the bacterial populations was observed on plates containing 1.25mg%, 2.5mg%, 3.75mg% sodium azide, beyond which the azide had lethal effect. These three corresponding mutants viz. S1, S2 and S3 had distinctly different phenotypic characteristics, e.g. loss of spore bearing mycelium and brown pigmentation with more brittle, irregular and diminished colony size compared to that of master strain, S0. Among the test bacteria, B. subtillis, a food poisoning bacterium, was found to be markedly susceptible to S2 and S3 mutants, while S. aureus was slightly sensitive compared to the S0 (Table-1).

The genomic DNA from the corresponding Streptomyces strains was isolated5 and a primer (sequence, CTGGCGTGAC; GC%, 70; mp.,34oC) was used for Randomly Amplified Polymorphic DNA (RAPD)-Polymerase Chain Reaction (PCR)6 to observe genetic polymorphisms. PCR reactions (30mL each) were performed in 1XPCR buffer (1.5mM MgCl2) with pH 8.3; dNTPs (2.5mM each), Taq-polymerase (1U) (obtained from GENEI Company, Bangalore, India), DMSO (1%), 10-20ng of template DNA and primers (10mM, obtained from University of British Columbia). The thermal cycler programme was set as: initial denaturation at 95 oC for 5min; 39 cycles of denaturation at 94oC for 1 min, annealing at 36 oC for 2min and extension at 72oC for 3min; followed by final extension at 72oC for 10min.

Table-1: Primary screening of antibacterial activities
of the Streptomyces strains (as length of inhibition, mm)

Fig 1: RAPD-PCR band pattern of Streptomyces
(λ DNA/Hind III marker, M)

Compared to S0, with five bands on 1% agarose gel electrophoresis (1322, 681, 596, 307, 206bp), two bands (1322 and 681) for mutant S3 were missing (Figure 1), which might be due to mutation in the primer binding region(s) ensuring no amplification of the products. Difference in morphology and antibacterial activity in S2 compared to those of the S0 could not be revealed in PCR ,possibly because mutations might have occureed outside primer binding regions necessitating use of other primer(s). Results indicate that using sodium azide as a mutagen, the antibacterial activities of Streptomyces spp. can be enhanced. This preliminary research, therefore, suggest the possibilities of strain improvement which may have wider applications in the modern chemotherapy .

Reference:
1. Williams ST, Sharpe ME, Holt JG. Streptomyces and related genera. In Bergey’s Manual of Systematic Bacteriology 1989; 4. Williams and Wilkins, USA.
2. Feldman SP, Mertelsmann R, Venuta S, Andreeff M, Welte K, Moor MAS. Sodium azide enhancement of Interleukin-2 production. Blood 1983; 61: 815-8.
3. Sano I, Mizumoto A, Sakai T, Tamura T, Itoh Z. Sodium azide induces relaxation of the canine gastric body by activating a guanylate cyclase dependant pathway. Neurogastroenterol Monit 1997; 9: 193-201.
4. Tamrakar R. Antibacterial activities of actinomycetes isolated from soils of Kathmandu valley. Thesis submitted to the Central Department of Microbiology, Tribhuvan University 1997.
5. Sambrook J, Fritsch t, Maniatis J. Molecular Cloning- A Laboratory Manual, 2nd Ed. Cold Spring Harbour Laboratory Press, London 1989.
6. Yu K, Pauls KP. Optimization of the PCR program for RAPD analysis. Nucleic Acid Res 1992; 20: 2606.

Strong mosquitocidal Bacillus thuringiensis from Mt. Everest

2007-06-11T06:40:00.000-07:00

Strong mosquitocidal Bacillus thuringiensis from Mt. Everest

Upendra Thapa Shreshtha¹, Kiran Babu Tiwari¹, Anjana Singh², Vishwanath Prasad Agrawal¹*

¹Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Universal Science College, Maitidevi, Kathmandu, Nepal; ²Central Department of Microbiology, Tribhuvan University, Kirtipur, Nepal

*Address for Correspondence: Prof. Vishwanath Prasad Agrawal, RLABB, Tel: +977-1-4442775, E-mail: vpa@wlink.com.np

Microbial insecticides are especially valuable as their toxicity to non-target animals and humans is extremely low compared to other commonly used chemical insecticides. They are safer for both the pesticide users and consumers of pesticide treated crops (Neppl, 2000). The soil bacterium Bacillus thuringiensis fulfills the requisites of a microbiological control agent of agricultural pests and vectors that cause massive crop destruction (Ben-Dov et al, 1999). The main target pest of B. thuringiensis insecticides includes various Lepidoptera (butterfly), Diptera (flies and mosquitoes), Coleopteran (Beatle) and some strains of nematodes (Schnepf et al, 1998). The kurstaki HD1 strain of B. thuringiensis is shown to possess wide spread insecticidal properties (Dulmage, 1970).

To study B. thuringiensis population from high altitude, soil samples were collected from Sagarmatha National Park (SNP) and Phereche of Khumbu region situated in the base camp of Mt. Everest, and processed in Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB). B. thuringiensis strains were isolated from soil samples by acetate selection method (Travers et al, 1987, Shrestha et al. 2006) and identified by standard microbiological techniques including colonial and morphological characteristics, and biochemical tests (Bergey’s Manual, 1986).

Out of 86 δ-endotoxin positive isolates (total 109), 10 randomly selected ones were used for insect bioassay. Larvae, collected from the ditches in local area of Bode, Bhaktapur, Nepal, were reared in a jar containing 100 ml of sterilized water containing 0.3 ml of 5% Brewer's Yeast and 5 ml of B. thuringiensis stationary phase culture and allowed to stand for 3 days. Although all isolates tested were effective against the larvae, isolate S₆ was the most effective of all (Fig. 1).

The isolates, obtained from the soil samples above 4000m altitudes where mosquitoes are not expected, may be novel. The S₆isolate showing potent insecticidal property tested against dipterans need to be studied further in larger trials so that it can have applicability to reduce the mosquitoes and different diseases caused by these vectors (Malaria, Filaria, Kalazar etc).

Acknowledgment : We thank CNR of Italy to support this work.

References:

Ben-Dov E, Wang Q, Zaritsky A, Manasherob R, Barak Z, Schneider B, Khamraev A, Baizhanov M, Glupov V, Margalith Y. Multiplex PCR screening to detect cry9 genes in Bacillus thuringiensis strains. Appl Environ Microbiol 1999; 65: 3714-6.

Bergey’s Manual of Systematic Bacteriology, Volume 2, 1986.

Dulmage HT. Production of spore-delta-endotoxin complex by variants of Bacillus thuringiensis in two fermentation media. J Invertebr Pathol 1970; 16: 385-9.

Neppl CC (2000). Managing Resistance to Bacillus thuringiensis Toxins. Environmental Studies University of Chicago.

Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR and Dean DH. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 1998; 62: 775-806.

Shrestha UT, Sahukhal GS, Pokhrel S, Tiwari KB, Singh A and Agrawal VP. Delta- endotoxin immuno cross-reactivity of Bacillus thuringiensis isolates collected from Khumbu base camp of Mount Everest region. J Food Sci Technol Nepal 2006; 2: 128-131.

Travers RS, Martin PA and Reichelderfer CF. Selective Process for Efficient Isolation of Soil Bacillus spp. Appl Environ Microbiol 1987; 53: 1263-6.

Enhanced Antibacterial Activity of Sodium Azide Treated Mutant Streptomyces Strain

2007-06-10T22:56:00.000-07:00

STUDY OF GENETIC POLYMORPHISMS AMONG STAPHYLOCOCCUS AUREUS RESPONDING VARIOUSLY TOWARDS VANCOMYCIN

2007-06-10T22:23:00.000-07:00

VRSA Proposal

STUDY OF GENETIC POLYMORPHISMS AMONG STAPHYLOCOCCUS AUREUS RESPONDING VARIOUSLY TOWARDS VANCOMYCIN

By: Kiran Babu Tiwari
Research Scientist
Research Laboratory for Agricultural Biotechnology and Biochemistry
email: kiranbabu.babukiran@gmail.com

1) Rationale:
a) Vancomycin is the terminal antibiotic for MRSA
Staphylococcus aureus is one of the most common causes of nosocomial infections, especially pneumonia, surgical site infections, and bloodstream infections. This bacterium has the ability to rapidly acquire antimicrobial resistance. Most S. aureus strains (>90%) are resistant to penicillin, and since the 1980s, methicillin-resistant S. aureus (MRSA) strains have become endemic in hospitals worldwide . One decade later during the 1990s, S. aureus isolates with diminished susceptibility to vancomycin (vancomycin-intermediate S. aureus [VISA]) were reported (Bozdogan et al., 2004; Avison et al., 2002; Bhateja et al., 2005). Glycopeptides, such as vancomycin, are frequently the antibiotics of choice for treatment of infections caused by the now common methicillin-resistant Staphylococcus aureus (MRSA). Incidences of vancomycin resistance in S. aureus (VRSA) have been increasing worldwide for the last 5 years. (Avison et al., 2002) The first clinical VRSA, Mu50, was isolated in Japan in 1997. Glycopeptides, such as vancomycin, are often the therapeutic drugs of choice for serious MRSA infections. However, failures of vancomycin therapy against S. aureus, due to the emergence of strains that are significantly less susceptible to vancomycin [vancomycinresistant S. aureus (VRSA)], are now well established (Avison et al., 2002). Recent reports of three S. aureus clinical strains with the vanA gene open a new era in staphylococcal antibacterial resistance (2-4). This latter development limits further potentially therapeutic options against these strains. (Bozdogan et al., 2004).

b) Mechanism of vancomycin resistance is simple mechano-biochemical reorganization of peptidoglycan biosynthesis
A thickened cell wall is responsible for the vancomycin resistance of VRSA strain Mu50, however, the mechanism of vancomycin resistance in other VRSA strains remained unclear (Cui et al., 2003). Thickening of the cell wall is a common phenotype of clinical VRSA strains and may be a phenotypic determinant for Vancomycin resistance in S. aureus. (Cui et al., 2003). VRSA strain Mu50 produces excessive amounts of peptidoglycan to make the thickened cell wall which become thinner with the loss of vancomycin resistance during drug-free passages and again became thick in the resistant mutant strains (Cui et al., 2003). The differences in the cell wall thickness between VRSA and passage-derived strains, vancomycin-resistant mutant strains and passage-derived strains, and VRSA and control strains were all statistically significant (P < 0.001)(Cui L et al., 2003). Vancomycin binds to the stem peptide of the membrane-anchored murein monomer (lipid II) at its Lys-d-Ala-d-Ala residue and inhibits the transglycosylation and transpeptidation reactions, preventing incorporation of the precursors into the bacterial cell wall (Périchon et al., 2004). The amount of glutamine-non-amidated muropeptide subunits (i.e. those containing d-glutamate rather than d-glutamine) in the Mu50 cell wall increases, and non-amidated muropeptides are poorer substrates for transpeptidases than amidated ones, although this is yet to be confirmed. Cell wall thickness and cross-linking result in vancomycin resistance is that the modified cell wall binds more vancomycin, due to the increased amount of terminal d-alanyl-d-alanine dipeptide (Avison et al., 2002). And thus the thickened cell wall not only traps a greater number of vancomycin molecules but also significantly reduces the time that vancomycin completely inhibits peptidoglycan synthesis (Cui L et al., 2003). The fine structure of the Mu50 cell wall is similar to that of an MRSA strain such as N315, except that the Mu50 peptidoglycan chains show significantly less cross-linking, and an increased content of pentapeptide chains (Avison et al., 2002). However, the resistance in enterococci (VRE) is due to synthesis of VanA-type glycopeptide, a modified precursors, ending in d-Ala-d-lactate (d-Ala-d-Lac) in place of d-Ala-d-Ala that result in 1,000-fold-lower affinity for glycopeptides (Périchon et al., 2004).

c) Development of resistance in the hospital ecological environment is, hence, likely to be faster
Isolates of vancomycin resistant S.aureus have emerged in many parts of the world. These isolates appear to achieve clinically relevant levels of resistance to vancomycin that leads to treatment failure (Bhateja et al., 2005). At present, the proportion of MRSA with reduced susceptibility to vancomycin is well known. Only 21 strains have so far been reported in literature, the first VRSA and hVRSA reported from Japan were MU 50 and MU 3, respectively. (Bhateja et al., 2005). Thus, the passage-derived strains with decreased MICs of vancomycin are best explained by the mechanism of heterogeneous resistance. The hetero-VRSA is the precedent strain of VRSA. Emergence of VRSA would be the result of vancomycin selection exerted upon a hetero-VRSA strain in the hospital, and the strain would return to hetero-VRSA status when vancomycin is not used for a while and its selective pressure lifted (Cui et al., 2003). Beta-lactam antibiotics are suspected to play a role in the dissemination of hetero-VRSA. In Japan, hetero-VRSA strains were found quite frequently in clinical isolates in the late 1980s before the introduction of vancomycin (vancomycin was introduced in 1991, and teicoplanin was introduced in 1998). This indicates that the conversion of VSSA to hetero-VRSA can be achieved in association with hetero- to homoconversion of methicillin resistance caused by beta-lactam selection (Cui et al., 2003). This suggests that the use of beta-lactam antibiotics for MRSA infection is a risk factor for the emergence of hetero-VRSA, although the precise genetic mechanism remains to be clarified. (Cui et al., 2003). The unusual feature of cell wall physiology of the vancomycin stressed S. aureus makes the culprit to develop resistance easily and faster. This may challenge the clinical microbiologist more than any other antibiotics used and practiced previously.

d) Early detection of the pathogen is of utmost importance
Emergence of a truly vancomycin-resistant S. aureus seriously threatens the most important treatment option available to clinicians for infections resulting from methicillin-resistant S. aureus. In the absence of vancomycin pressure, vancomycin resistance was found to be unstable and expressed at a low level. This low-level expression of vancomycin resistance in S. aureus may be the reason why these strains are hard to detect clinically (Bozdogan et al., 2004). So, early detection of the pathogen is of utmost importance.

e) Many controversies about the method standardization for MIC values and interpretations
Vancomycin resistance can be difficult to detect in clinical microbiology laboratory. Disk diffusion sensitivity testing by standard 30µg vancomycin frequently misclassifies intermediately susceptible isolates as fully susceptible. Presently MIC determinations by broth or agar dilution or by E test are the gold standard for determining vancomycin susceptibility, but these methods are not suitable for routine use in the diagnostic laboratories (Bhateja et al., 2005).Vancomycin resistance in S.aureus is difficult to define mainly because of methodological problems in their detection (Bhateja et al., 2005). No simple correlation between glycopeptide and beta-lactam MICs was seen, while significant correlations between MICs of vancomycin and teicoplanin (r = 0.679; P < r =" 0.787;"> 32µg/mL are resistant (Bhateja et al., 2005). Thus these observations lead to difficulty in interpretations.

f) Genetic analysis for Vancomycin resistance:
Complex mechanisms producing changes in cell wall content and composition generate the VRSA phenotype, but the genetic basis of these changes has not yet been determined (Avison et al., 2002). In 2002, the first two clinical isolates of vancomycin-resistant Staphylococcus aureus (VRSA) possessing acquired vanA operon containing Tn1546 were recovered in Michigan and Pennsylvania (Clark et al., 2005) on conjugative plasmids with a broad host range in Gram-positive bacteria. (Bozdogan et al., 2003). The first two VRSA isolates arose from independent genetic events (Clark et al., 2005). The gene clusters are not stable genetic elements and serial passage of the strain in the absence of vancomycin led to loss of the vanA cluster and, consequently to restoration of vancomycin susceptibility (Bozdogan et al., 2003).

VanA-type resistance is characterized by high-level inducible resistance to both vancomycin and teicoplanin and is mediated by Tn1546 or closely related elements. Tn1546, which belongs to the Tn3 family of transposons, is composed of seven genes, two of which encode a transposase and a resolvase responsible for the movements of the elements of the vanA operon, and is delineated by imperfect inverted repeats. The vanH, vanA, and vanX genes code for proteins that are necessary for the expression of resistance. VanH is a dehydrogenase that converts pyruvate to d-Lac, VanA a ligase that uses d-Lac and a d-Ala residue to synthesize the depsipeptide d-Ala-d-Lac, which is incorporated into the peptidoglycan precursors, and VanX is a d,d-dipeptidase that hydrolyzes the dipeptide d-Ala-d-Ala formed by the endogenous Ddl chromosomal d-Ala:D-Ala ligase, thus reducing the level of normal peptidoglycan precursors ending in d-Ala-d-Ala. The VanY d,d-carboxypeptidase, not essential for resistance, cleaves the d-Ala C-terminal residue of pentapeptide precursors synthesized by using d-Ala-d-Ala dipeptides that have escaped VanX hydrolysis. The vanZ gene is implicated in teicoplanin resistance by an unknown mechanism. Expression of the vanA operon is regulated by two genes, vanR and vanS, located upstream from vanH that form a two-component regulatory system (Périchon etb al., 2004).

It is difficult to rationalize a role for these mutations in the VRSA phenotype. It is well documented that Mu50 grows more slowly than N315, which may be explained by the loss of such important functions. It has been hypothesized that the basis of the VRSA phenotype in Mu50 is an increased level of peptidoglycan biosynthesis, resulting in a considerably thicker cell wall. In addition to this, there is a reduced level of glutamate amidation in the cell wall, which may be responsible for reduced cross-linking and the reduced ability of d-d-carboxypeptidases to act on the peptidoglycan network, conserving d-alanyl-d-alanine terminal dipeptides, which bind and sequester vancomycin (Avison et al., 2002).

Our analysis has identified loss-of-function mutations in genes encoding enzymes responsible, in part, for the committed stage of peptidoglycan biosynthesis and for the manufacture of intermediary metabolites that are precursors of glutamine, and hence glucosamine. These findings will inform future targeted study of the biochemistry of vancomycin resistance in S. aureus (Avison et al., 2002).

To facilitate the genetic investigation, entire genome sequences of the archetypal VRSA (Mu50), and vancomycin-susceptible MRSA strains N315, EMRSA 16 and COL were compared. The in silico analysis revealed several loss-of-function mutations in Mu50, affecting important cell wall biosynthesis and intermediary metabolism genes, not previously reported. The new findings provide further evidence for the hypothesis that vancomycin resistance in Mu50 is due to fundamental changes, important to metabolic pathways that impinge on peptidoglycan biosynthesis (Avison et al., 2002).

The complete sequence flat-files of the genomes of Mu50 and N315 were using a specially written macro that eliminates all non-amino acid sequence information to produce a ‘proteome’ for each genome for all predicted open reading frames (ORFs) joined end to end. 164 individual differences between the proteomes of Mu50 and N315 were found. Of these, 114 are minor, i.e. single amino acid substitutions, and their effects cannot be predicted, though it cannot be assumed that they are neutral. Accordingly, each product would have to be investigated individually. DNA sequences for the remaining 50 Mu50 ORFs showing more marked differences from those in N315 were obtained. In 13 instances, the sequence in N315 was different from those in the other comparators (genome sequence data from MRSA COL), so these changes could not be linked to the VRSA phenotype. Where the Mu50 genomic copy was found to differ from those in the three MRSA strains (37 instances), many of the changes were located in ‘hypothetical’ ORFs, and genes clearly unrelated to vancomycin resistance (e.g. endotoxin genes and antigenic determinants, etc.). When these are eliminated, a total of 17 loss-of-function mutations specific to the Mu50 genome in genes encoding characterized functions were identified. A link between five of these disruptions and the biochemical differences that have been previously noted between Mu50 and MRSA strains is compelling (Avison et al., 2002).

The authors explained the complementary genes and functions for those ones that seemed to be critical to cell wall physiology related to vancomycin stress, eg., (a) murZ for murA that encodes UDP-GlcNAc-enolpyruvyl transferase, mrp for fmtB homologue, and (b) 2-ketoglutarate oxidoreductase and succinyl-CoA synthase for 2-ketoglutarate dehydrogenase (Kdh, odhA) and succinate dehydrogenase (Sdh, sdhB) respectively (Avison et al., 2002). Comparison of the PFGE patterns of the parent and passage-derived strains demonstrated that 13 of the 16 pairs shared identical banding patterns (Cui L et al., 2003). Avison et al. (2002) pointed out that Glutamine synthetase (GS) and glucosamine synthase activity in Mu50 is higher than in N315 suggesting that Mu50 is geared to direct more carbon into synthesis of GlcNAc. One side-effect of hyperproduction of GlcNAc would be a shrinking of the l-glutamine pool, because it would be used faster to produce glucosamine. This, in turn, would mean that the amount of l-glutamine available for amidation of d-glutamate in cell wall precursors would be reduced. The observed disruption of the genes odhA and sdhB, if confirmed in other VRSA strains, does fit with the hypothesis that perturbation in this branch of intermediary metabolism is important in the VRSA phenotype of Mu50 (Avison et al., 2002).

The analysis is necessarily limited in a number of ways. There may well be other DNA sequence differences in Mu50 that do not cause coding changes but do influence susceptibility to vancomycin, such as mutations in promoter regions or transcription regulatory sites that affect the expression of relevant genes. The analysis of global gene expression in VRSA strains is in progress, although limited analysis of Mu50 has yielded some interesting results. A major limitation is that, of necessity, the genetic analysis only relates to one VRSA strain, Mu50. An investigation to determine whether mutations similar to those found in Mu50 are also present in other clinical VRSA strains is underway (Avison et al., 2002).

g) RAPD-Typing:

With these views, a deliberate search should be done to find out the different sites in the chromosome of the hVISA (hetero-vancomycin intermediately resistance S. aureus), VISA (vancomycin intermediately resistance S. aureus) and VRSA (vancomycin resistant S. aureus) strains that differed with that of VSSA (vancomycin susceptible S. aureus) / MRSA (Methicillin resistance S. aureus). A series of RAPD primers can be used to locate the differences among the strains. A concurrent data is expected at least from some primers, and the selected primers can be used to predict the possible hVISA, VISA and VRSA strains.

2) Hypothesis: VSSA and VRSA have same genomic compositions

3) Procedure: (As described elsewhere)

a) Isolation of staphylococci from clinical samples collected in the Tertiary Care hospital in Nepal
b) Biochemical characterization
c) Transportation of the isolates to RLABB d) MIC determination and characterization into VSSA, hVISA, VISA and VRSA
e) Isolation of whole DNA and/or chromosomal DNA and plasmid DNA
f) Primer selection for reproducible genetic polymorphisms
g) RAPD-PCR
h) Interpretation

4) Expected outcome:
a) Different colonial morphologies
b) Different biochemical patterns
c) MIC values for VSSA, hVISA, VISA and VRSA
d) Different RAPD-PCR patterns

5) References

Bozdogan B, D Esel, C Whitener, F A. Browne and Pr C. Appelbaum (2003) Antibacterial susceptibility of a vancomycin-resistant Staphylococcus aureus strain isolated at the Hershey Medical Center Journal of Antimicrobial Chemotherapy 52, 864-868.

Cui L, X Ma, K Sato, K Okuma, F C. Tenover, E M. Mamizuka, C G. Gemmell, Kim, M-C Ploy, N. E Solh, V Ferraz, and K Hiramatsu 2003 Cell Wall Thickening Is a Common Feature of Vancomycin Resistance in Staphylococcus aureus J Clin Microbiol. January; 41(1): 5–14.

Clark N C, L M. Weigel, J B. Patel, and F C. Tenover 2005 Comparison of Tn1546-Like Elements in Vancomycin-Resistant Staphylococcus aureus Isolates from Michigan and Pennsylvania Antimicrob Agents Chemother; 49(1): 470–472.

Bozdogan B, L Ednie, K Credito, K Kosowska, and P C. Appelbaum 2004 Derivatives of a Vancomycin-Resistant Staphylococcus aureus Strain Isolated at Hershey Medical Center Antimicrob Agents Chemother. 48(12): 4762–4765.

Périchon B and P Courvalin 2004 Heterologous Expression of the Enterococcal vanA Operon in Methicillin-Resistant Staphylococcus aureus Antimicrob Agents Chemother. 48(11): 4281–4285.

Avison M B , P M. Bennett, R A. Howe and T R. Walsh 2002 Preliminary analysis of the genetic basis for vancomycin resistance in Staphylococcus aureus strain Mu50 Journal of Antimicrobial Chemotherapy 49: 255-260.

Bhateja P, M T Pandya, M Fatma, T R Ashok 2005 Detection of vancomycin resistant Staphylococcus aureus: A comparative study of three different phenotypic screening methods Indian Journal of Medical Microbiology 23( 1): 52-55.

2007-06-10T04:11:00.000-07:00

SDS PAGE

2007-06-07T19:16:00.000-07:00

How dose an SDS-PAGE really work?

HOW DOES AN SDS PAGE GEL REALLY WORK? 06/06/2007

The system most people use for separating proteins by gel electrophoresis was formulated by Laemmli (Nature 227:680-685 [1970]). It is such a commonly used laboratory technique that nowadays we take it for granted and I dare say that many of us don’t know how a discontinuous pH gel system actually works.

First the sample. Most SDS PAGE sample buffers contain the following: SDS (sodium dodecyl sulphate, also called lauryl sulphate), b-mercaptoethanol (BME), bromophenol blue, glycerol, and Tris-glycine at pH 6.8. BME is added to prevent oxidation of cysteines and to break up disulfide bonds. Bromophenyl blue is a dye that is useful for visualizing your sample in the well and tracking its progress through the gel. Glycerol is much more dense than water and is added to make the sample fall to the bottom of the sample well rather than just flow out and mix with all the buffer in the upper reservoir. The interesting components are the buffer and the SDS.

SDS is an ionic detergent that binds to the vast majority of proteins at a constant ratio of 1.4 gm SDS/gm protein. A few proteins like tubulin do not bind at this ratio and this is one reason why some proteins migrate anomalously (there are other reasons as well so you shouldn’t put too much faith in the apparent molecular weight estimated from an SDS PAGE gel). Since SDS is an anionic detergent it imparts a negative charge to all the proteins in your sample. More importantly, these charges swamp the inherent charge of the proteins and give every protein the same charge-to-mass ratio. Because the proteins have the same charge-to-mass ratio, and because the gels have sieving properties, mobility becomes a function of molecular weight. But what about running gels, stacking gels, electrode buffer, and all these different pHs?

The velocity of a charged particle moving in an electric field is directly proportional to the field strength and the charge on the molecule and is inversely proportional to the size of the molecule and the viscosity of the medium. Adding a gel with sieving properties (that is a gel where the resistance to the motion of a particle increases with particle size) increases the differences in mobility between proteins of different molecular weights. This is the basis of separation. The problem now becomes how to line up all the proteins in an orderly fashion at the starting gate. That’s where the discontinuous pH part comes in.

Laemmli gels are composed of two different gels (stacker and running gel), each cast at a different pH. In addition, the gel buffer is at a third, different pH. The running gel is buffered with Tris by adjusting it to pH 8.8 with HCl. The stacking gel is also buffered with Tris but adjusted to pH 6.8 with HCl. The sample buffer is also buffered to pH 6.8 with Tris HCl (note all the chloride ions – they will become important in a minute). The electrode buffer is also Tris, but here the pH is adjusted to a few tenths of a unit below the running gel (in this case 8.3) using only glycine – nothing else. We run our gels at constant voltage.

So here’s what happens when you turn on the power. Glycine is a weak acid and it can exist in either of two states, an uncharged zwitterion, or a charged glycinate anion (that is to say, negatively charged). At low pH it is protonated and thus uncharged. At higher pH it is negatively charged. When the power goes on the glycine ions in the running buffer want to move away from the cathode (the negative electrode) so they head toward the sample and the stacking gel. The pH there is low and so they lose a lot of their charge and slow down. Meanwhile, in the stacker and sample the highly mobile chloride ions (which are also negatively charged) move away from the cathode too. This creates a narrow zone of very low conductance (in other words very high electrical resistance) in the top of the stacking gel. Because V=IR almost all of the voltage that you put across the gel (110 Volts is typical for stacking) is concentrated in this small zone. The very high field strength makes the negatively charged proteins move forward. The trick, however, is that they can never outrun the chloride ions. If they did they would find themselves in a region of high conductance and very low field strength and would immediately slow down. The result is that all the proteins move through the stacker in a tight band just behind the moving front of chloride ions. Behind them, the pokey glycine ions straggle along as best they can (they do move, but with lower mobility than the chloride ions).

The effect of this moving zone of high voltage is that all the proteins reach the running gel at virtually the same time so that migration of the proteins is truly a function of molecular size and not some complicated function of how carefully you loaded the gel and when you started the voltage.

When the big caravan of ions hits the running gel everything changes. The pH goes way up and the glycine becomes deprotonated (and thus more negatively charged). The mobility of the glycine goes way up and the mobility of the proteins goes way down (due to the sieving properties of the gel). The result is that the glycine races past the protein and the proteins are no longer in a narrow zone of very high resistance (and very high electric field). They find themselves in a much more relaxed, uniform electric field where they can chill out a bit. Move at their own pace.

Tips for running good gels:

1. After pouring the running gel, carefully overlay it with ethanol or another imiscible liquid. This will give you a nice flat surface. Also, since polymerization of acrylimide is inhibited by oxygen it will speed up polymerization.
2. For the mini-gels we run the minimum protein loading per well (single band) is 0.1 µg for standard Coomassie staining and 2 ng for silver staining. I haven’t tested it but my impression is that Simply Blue staining is within a factor of two as sensitive as standard Coomassie staining.
3. The maximum protein loading per well (for a mixture of proteins of different sizes) is about 40 µg. If you exceed amount this your gel will look like crap.
4. KCl causes SDS to precipitate. If you samples contain KCl you should dilute them or methanol precipitate them and resuspend them in 1X sample buffer. With low concentrations of KCl (<200 mM) you can run them on the gel but you should loaed every lane with sample buffer containing the same concentration of KCl (even if they are blanks). This will help the gel run a little less anomalously.
5. If your sample buffer turns yellow, it is at the wrong pH. Add NaOH or

Safety Notes:

- Acrylimide is extremely toxic, causing central nervous system paralysis. It can be absorbed through unbroken skin. If skin comes in contact with acrylimide solution or powder, wash immediately with soap and a lot of water. Unpolymerized acrylimide should be polymerized with excess catalyst and disposed of with solid waste. DO NOT POUR UNPOLYMERIZED ACRYLIMIDE DOWN THE SINK.
- Amonium Persulfate should be made up fresh or used from a relatively fresh stock. It goes bad after a week or two in the refrigerator. It can be disposed of by dilution with water and pouring down the sink.
- TEMED should be stored in the refrigerator in dark glass bottles. A bottle should be good for about a year, maybe longer.

Posted by prabhat

Announcement

2007-06-06T05:33:00.000-07:00

Research Laboratory for Agricultural Biotechnology and Biochemistry ( RLABB ) was established by Prof. Vishwanath P. Agrawal in 1986. It is based in Kathmandu, Nepal. This year its name was shortened to Research Laboratory for Biotechnology and Biochemistry because its research activities have been diversified to cover the important field of microbiology. It has been registered as a non - profit organization under the company act of Nepal Govt.

I request all to view our postings regularly. If you want to contribute to this blog, please let me know and then I will invite you. Thank you.

Prof. Vishwanath P. Agrawal
Executive Director
RLABB