Cloning of Bacillus thuringiensis cry3 fragment in Escherichia coli

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).

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