Cloning of cry3 fragment in Escherichia coli

Shyam K. Shah1, Kiran Babu Tiwari1,2, Upendra Thapa Shrestha2, Subarna Pokhrel3, Eitan Ben-Dov4 and Vishwanath P. Agrawal1,2*
(1) Department of Biochemistry, Universal Science College, Pokhara University, Kthmandu, Nepal;
(2) Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal;
(3) Department of Enzyme Engineering, Seoul National University, Korea;4Department of Life Sciences, Ben-Gurion University of the Negev, Be’er-Sheva 84105, Israel*

Correspondence Address: Dr. Vishwanath P. Agrawal, Professor of Biochemistry, Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal.Email: vpa@wlink.com.np. Contact: +977-1-2110043

ABSTRACT

Bacillus thuringiensis was isolated and purified from the soil sample collected from Khumbu, Mt. Everest base camp. Total DNA was extracted and PCR was done using nine universal primers for cry1 to cry9. A specific band of about 300bp was amplified with universal primer 3. DNA library was created by digesting the DNA with HindIII, ligation into pUC18 followed by transformation of Escherichia coli HB101. Using universal primer 3 for PCR, 100 out of 1000 clones prepared were screened and three were found to posses cry3 specific fragment of the same size as before. The cry3 specific fragment was cloned, extracted, purified and sent for sequencing. As the bacterium was isolated from high altitude, the gene may be novel with promising for biological control and management of insects.

INTRODUCTION

Bacillus thuringiensis is an aerobic, ubiquitous, gram positive, spore forming bacterium that forms an insecticidal parasporal crystal protein (δ-endotoxin). The crystal protein can be used to control certain insect species among the orders lepidoptera, diptera, coleopteran (Beegle and Yamamoto, 1992) and, hence, is a useful alternative to synthetic chemical pesticide applied in commercial agriculture, forest management and mosquito control. The genes encoding δ-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 δ-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 swell and lyses. Recently, the crystal proteins and their genes have been classified based on their structure, antigenic properties and activity spectrum.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 has to be explored yet, especially from extreme environments in order to find novel strains. To study B. thuringiensis population from high altitude, soil samples were collected from Khumbu, Mt. Everest base camp and the bacteria were isolated in Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB). Further, biochemical and insecticidal properties had already done in RLABB.

MATERIALS AND METHODOLOGY

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

Isolation of total DNA: DNA from Bacillus thuringiensis were isolated by incubating a 50 ml Bacillus thuringiensis culture overnight at 37°C in LB medium with vigorous shaking. Cells were pelleted by centrifugation for 5 min at 8000 rpm and resuspended in sterile SSC buffer. Then cells were lysed using lysozyme (10mg/ml) and Sodium deducible sulphate (10%). DNA was extracted by phenol: chloroform extraction method (Sambrook et al 1989). Then all DNAs preparations were purified by gel extraction method using Kit (Genei, Banglore ,India).

PCR using universal primers (Ben-Dov et al 1997): Universal primers for cry1 (Un1), cry2 (Un2), cry3 (Un3), cry4 (Un4), cry5 (Un5), cry6 (Un6), cry7 (Un7), cry8 (Un8) and cry9 (Un9) were used for PCR. Amplification was carried out in a DNA programmable Thermal controller (MJ Research, Inc.) for 35 reaction cycles. Each reaction mixture was carried out in 50µl system containing 2µl of template DNA mixed with 5µl of 10X reaction buffer, (2.5mM) dNTPs, 10 pmole each primer and 0.5U/µl of Taq polymerase (Genei, Banglore, India).Template DNA was denatured for 1 min at 94°C and annealed to primers at 50°C for 1 min 30s. Extension of PCR products were achieved at 72°C for 2 min. The PCR products were analyzed by 2% agarose gel electrophoresis.

Restriction digestion and ligation of Bt DNA and vector (pUC18): The vector was restricted by 2.5 µl of HindIII (5U/µl) with 10µl of 10X buffer in 100 µl digestion mixture and dephosphorylated with 5U of Calf-intestinal alkaline phosphatase. Similarly, 40 µl of Bt DNA was digested with 5 µl of HindIII (5U/µl) with 10 µl of 10X buffer C in 100 µl digestion mixture(Sambook et al 1989). The vector and inserts were purified using DNA purification kit and then ligated (50ng of vector and 257.078 ng of inserts using 0.5U/µl of T4 DNA ligase in a volume of 10µl) (Johnson et al 1996).

Transformation: Transformation was carried out as described by (Schnepf and Whiteley 1981). E. coli HB101 transformants were selected on media containing ampicillin at 100µg/ml.

Screening of bacterial colonies: Individual transformant was mass cultured in 5ml LB broth and plasmid was extracted by alkaline extraction procedure (Birnboim and Doly, 1979). The recombinant plasmids were screened through PCR method using universal primer 3 (Un3) as described above.

Isolation and purification cry3 fragment: The cry3 fragment (~300bp) was amplified by PCR and electrophoresed (2% agarose gel). The product was extracted, purified and stored in deep freeze till sending for sequencing.

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). A total of 1000 individual E. coli chimeras were isolated and stored in deep freeze. So far 100 clones were screened and three were found to have cry3 specific fragment.

Fig. 1. Agrose 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 (λ DNA/HindIII); 1, Un1; 2, Un2; 3, Un3; 4, Un4; 5, Un5; 6, Un6; 7, Un7; 8, Un8 and 9, Un9.

Fig 2. Agarose (2%) gel electrophoresis of PCR products with Un3 primers. Template plasmids were from recombinant E. coli. Lanes: M, marker (λ DNA/HindIII); C, negative control; 1, plasmid containing cry3 specific fragment; 2, plasmid without cry3 specific fragment.

DISCUSSION:

The crystal proteins of B. thuringiensis have been extensively studied because of their pesticidal properties and their high natural level of production. The increasingly rapid characterization of new crystal protein genes has resulted in a 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 protein gene has not done yet in Nepal. Hence, this study was planned with a hope to find novel B. thuringiensis that are cold tolerant, as it was isolated from a high altitude soil samples (Khumbu, Mt. Everest base camp).The bacteria were found to possess cry3 gene as universal primer gave the specific amplification product on PCR among the nine universal primers tested. 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). 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. The amplified product was concentrated and purified. The product is being sent for sequencing to know whether it is novel or not. When found to be novel, a piece of the fragment can be used to synthesize specific primers and/or probe to detect the strain in the given population.

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