A DEMONSTRATIVE MANUAL FOR BASIC MOLECULAR BIOLOGY PRACTICAL

Saturday, July 14, 2007

A DEMONSTRATIVE MANUAL FOR BASIC MOLECULAR BIOLOGY PRACTICAL
A DEMONSTRATIVE MANUAL FORBASIC MOLECULAR BIOLOGY PRACTICALFOR THE STUDENTS OFDEPARTMENT OF MEDICAL MICROBIOLOGYNOBEL COLLEGEPOKHARA UNIVERSITYJul 1-6, 2007Prepared byKiran Babu TiwariAsst. Prof. of Microbiology, USCResearch Scientist, RLABBIn association withUpendra Thapa ShresthaNirajan BhattaraiVijayendra Agrawal(Research Scientists, RLABB)Reference:Professor Dr. Vishwanath Prasad AgrawalExecutive DirectorResearch Laboratory for Biotechnology and Biochemistry (RLABB)www.uscollege.edu.np/rlabbhttp://www.rlabb.com.np/http://www.vpagrawal.com/AcknowledgementWe are indebted toProf. Dr. Vishwanath P. Agrawal,Executive Director, Research Laboratory for Biotechnology and Biochemistry (RLABB);Director, Universal Science College; andAcademician, National Academy of Science and Technology (NAST)for providing the facilities to conduct the experiments enlisted in this manual.CONTENTSTITLE PAGEACKNOWLEDGEMENTCONTENTSJULY 1:
ORIENTATION ON BASIC MOLECULAR BIOLOGY PROCEDURESJULY 2:
EXPT 1. ISOLATION AND PURIFICATION OF BACTERIAL GENOMIC DNAEXPT 2. ISOLATION AND PURIFICATION OF RNA
JULY 3:
EXPT 3. ISOLATION AND PURIFICATION OF BACTERIAL PLASMIDS BY ALKALINE LYSIS METHOD
JULY 4:
EXPT 4. SPECTROPHOTOMETRIC ANALYSIS OF DNAEXPT 5. RESTRICTION DIGESTION OF DNAJULY 5:
EXPT 6. AGAROSE GEL ELECTROPHORESIS OF DNAJULY 6:
EXPT 7. POLYMERASE CHAIN REACTION
Day 1
ORIENTATION ON BASIC MOLECULAR BIOLOGY PROCEDURES1. Principle of nucleic acids isolation from bacteriaNucleic acids are present as nucleoprotein complexes in cells. The major problems encountered in isolation of pure and intact DNA or RNA molecules are: degradation of high molecular weight nucleic acids by mechanical damage or by hydrolytic action of nucleases, contamination of DNA preparations with RNA and vice versa and contamination of nucleic acids with proteins, polysaccharides and other high molecular weight compounds. Methods have been devised for isolation of nucleic acids from different sources taking adequate precautions to eliminate or minimize the above problems.The main steps involved in isolation of nucleic acids are:
(a) Disruption of cells: Disintegration of bacterial cells can be achieved by treating them with cell coat hydrolyzing enzyme, i.e. lysozyme in presence of a detergent. at low temperature in buffers containing EDTA (chelates Mg2+ ions which are required for DNase activity). For isolation of RNA, and inhibitor of RNase such as bentonite, diethylpyrocarbonate, placental RNase inhibitor etc. is included in the extraction buffer.
(b) Dissociation of nucleo-protein complexes: The approach employed is such that the proteins either get dissociated or degraded while nucleic acids remain unaffected and intact. This is generally achieved by using detergents like SDS, phenol or broad-spectrum proteolytic enzymes such as pronase or proteinase K. Alkaline pH and high concentration of salts improve efficiency of the process.
(c) Removal of contaminating materials and precipitation of nucleic acids: Proteins are removed by treatment with phenol or mixture of chloroform-isoamyl alcohol or phenol-chloroform. Upon centrifugation, the denatured proteins form a layer at the interface between upper aqueous and lower organic phases, lipids and other contaminants remain in the same organic phase while nucleic acids are recovered in the aqueous phase from which they are precipitated with ethanol. For isolation of DNA, the contaminating RNA is removed by selective salt precipitation or treatment with DNase-free RNase. Conversely while isolating RNA, the preparation is incubated with DNase for eliminating DNA as an impurity.
(d) General precautions while handling nucleic acids:1. All glasswares and solutions (except organic solvents) should be sterilized.2. Gloves should be worn to avoid contamination of the experimental material and apparatus with nucleases with occur in fair abundance in skin exudates.3. Phenol causes severe burns and phenol-containing solutions should, therefore, be handled with care. Thoroughly rinse the burns with large volume of water. Do not use ethanol.4. For work with RNA, rinse all glasswares with 1% diethylpyrocarbonate solution to inactivate RNase and then autoclave them.2. Basic components for Molecular Biology experimentsLB (Lauria-Bertani) broth: 0.5% NaCl, 1% Tryptone, 0.1% Yeast extractOvernight Log- phase cultureSolution I (Lysis buffer): 25mM Tris, 50mM Glucose, Lysozyme [10mg/ml stock; GNB (Gram Negative Bacteria) 0.5mg/ml, GPB (Gram Positive Bacteria) 3-5mg/ml], pH 8.0Solution II (Lysis): (a) for plasmid isolation: 0.2M NaOH, 10% SDS (b) for genomic DNA isolation: 10% SDS only.Proteinase K: 10mg/ml stock, 10-20mg/65°C/3hrsSolution III: 3M Sodium acetate, pH 4.8 by acetic acidPhenol: distilled, protein precipitantChloroform: protein precipitant, phenol solventRNase: 5mg/ml stock, 1-2mlAbsolute ethanol/Isopropanol: DNA precipitant, 2.5vol of ethanol or 1vol of isopropanol70% ethanol: washing DNA precipitantTE buffer: DNA resuspending buffer, 10mM Tris, 1mM EDTA, pH 8.0; Autoclave before useTAE buffer: Electrophoresis, 50X, Tris, 24.2gm; acetic acid, 5.71ml; EDTA (0.5M), 11.1ml; DW, 100ml; pH 8.0; Autoclave before useLoading dye: 6X; 50% Glycerol, 6.0ml; 2%BPB (Bromophenol Blue), 1.0ml; DW, 3ml; Always use sterile DWEthidiun Bromide (EtBr)*: 10mg/ml stock (Final concentration: 0.5mg/ml)λ/HindIII Marker: 23.13Kb, 9.42Kb, 6.56Kb, 2.32Kb, 2.07Kb, 0.56Kb and 0.13KbRestriction enzymes: EcoRI and HindIII (10U/ml each)Restriction enzyme buffers for EcoRI and HindIII (10X each)Sterile Double distilled water (DDW)Water bathCold centrifuge (upto 20000rpm)Micropipettes and sterile tips*Note: EtBr is carcinogen, so, handle with gloves
Day 2
Expt. 1. Genomic DNA extraction from bacteria-Take 1.5ml of overnight LB-broth culture of bacteria in a MFT (Microfuge tube) and spin at 5000rpm for 10min.
-Remove the supernatant and spin once with same volume as above to collect more cell mass.
-Remove the supernatant.
-Add 100µl Sol. I and keep for 30min at RT (Room temperature).
-Add 1/10 vol. of 10% SDS and swirl to mix.
-Add Proteinase K (1-2 µl) and incubate for 30min at 37ºC with gentle shaking.
-Add 1-2µl of RNase and incubate for 10min at RT.
-Add equal vol. of Phenol:Chloroform (1:1), mix gently and keep for 10min at RT.
-Centrifuge at 8000rpm for 10min at 4ºC and collect the supernatant in a new sterile MFT.
-Add equal vol. of 3M sodium acetate, mix and stand it for an hour in cold.
-Spin at 10000rpm at 4ºC for 15min and wash the pellet with 70% ethanol.
-Dissolve the pellet collected during spin in 50µl TE and store in deep freeze.Expt. 2. RNA extraction from bacteria
-Take 1.5ml of overnight LB-broth culture of bacteria in a MFT and spin at 5000rpm for 10min.
-Remove the supernatant and spin once with same volume as above to collect more cell mass.
-Remove the supernatant.
-Add 100µl Sol. I and keep for 30min at RT.
-Add 1/10 vol. of 10% SDS and swirl to mix.
-Add Proteinase K (2 µl) and incubate for 30min at 37ºC with gentle shaking.
-Add equal vol. of Phenol:Chloroform (3:1) solution and mix gently.
-Centrifuge at 8000rpm for 10min at 4ºC and collect the supernatant in a new sterile MFT.
-Add equal vol. of 3M sodium acetate and/or two vol. of absolute ethanol. Mix and stand it for 1-2 hour in cold.
-Spin at 15000rpm at 4ºC for 15min and wash the pellet with 70% ethanol.
-Dissolve the pellet collected during spin in 50µl TE (pH 7.0) and store in deep freeze.Day 3
Expt 3. Plasmid DNA extraction from bacteria-Take 1.5ml of overnight LB-broth culture of bacteria in a MFT and spin at 5000rpm for 10min.
-Remove the supernatant and spin once with same volume as above to collect more cell mass.
-Remove the supernatant.
-Add 100µl Sol. I and keep for 30min at RT.
-Vortex for a while and add Proteinase K (2µl) and incubate for 30min at 37ºC with gentle shaking.
-Add freshly prepared Sol. IIa (200µl) and mix gently (Do not vortex).
-Add ice cold Sol. III (150µl) and mix gently (Do not vortex).
-Add 1-2µl of RNase and incubate for 10min at RT.
-Add equal vol. of Phenol: Chloroform (1:1), mix gently and keep for 10min at RT.
-Centrifuge at 8000rpm for 10min at 4ºC and collect the supernatant in a new sterile MFT.
-Add equal vol. of isopropanol and stand it for an hour in cold.
-Spin at 13000rpm at 4ºC for 15min and wash the pellet with 70% ethanol.
-Dissolve the pellet collected during spin in 50µl TE and store in deep freeze.Day 4
Expt. 4. Spectrophotometric analysis of DNA1. Remove the DNA preparation from the freeze and thaw it.
2. Transfer 495μl TE buffer to a quartz cuvette and add 5μl of the DNA preparation. Mix well.
3. Set the spectrophotometer to 260nm and blank the instrument with TE buffer.
4. Measure the absorbance (A260) of the DNA dilution.
5. Repeat steps 3 & 4 with the spectrophotometer set at 280 nm.
6. Calculate the DNA concentration from A260. [µg/ml = A260 X dilution X 50].
7. Calculate A260/A280 in order to estimate the purity of the DNA preparation (Pure DNA has a A260/A280 ratio of 1.8 – 2.0).
Expt. 5. Restriction digestion of DNA1. Dilute the DNA extracts, Marker/s and enzymes in suitable solvents accordingly.
2. Calculate the volume for digestion reaction as shown in the table given below.Components............ Rxn. .........1 Rxn. 2λ DNA (1µg/µL) ............10 µL ..............-DNA extract (1µg/µL) ...- ....................10 µLHindIII (10U/µL) .........1 µL ................1 µLHindIII buffer (10X) ....1 µL ................1 µLDW ..................................8 µL ................8 µLTotal ...............................20 µL ..............20 µL3. Perform digestion reaction by mixing the components in sterile MFT.
4. Briefly centrifuge the contents and keep at 37ºC for 6hrs.
5. Perform agarose gel electrophoresis to interpret the results.Day 5
Expt. 6. Agarose electrophoresis of DNA1. Prepare 0.8% agarose gel in 1X TAE buffer (28ml).
2. Dissolve agarose completely in micro-oven and cool to 600C.
3. CAREFULLY, add EtBr into the gel solution to final concentration of 0.5µg/ml.
4. Position a comb in the mold. Pour into gel mold and let it cool for 30 minutes.
5. Pour the TAE buffer into the gel buffer reservoir.
6. Prepare sample taking 20µl of DNA sample and mix with 4µl blue juice.
7. Carefully remove the comb.
8. Load the DNA (15 µl) in the wells, flanking wells with similarly processed DNA size standard.
Note: the amount of the sample that can be loaded in a well depends on the thickness of the gel as well as dimensions and placing of the comb.9. Put the lid on the gel apparatus; attach the electrodes and adjust voltage to 100 volts.
10. Allow the gel to run until line of blue juice is visible near the end of the gel.
11. Turn off the current and visualize the gel in UV transilluminator.
12. Interpret the results.Day 6
Expt 7. RAPD-PC(Randomly Amplified Polymorphic DNA – Polymerase Chain Reaction)1. Thaw the DNA extract and dilute in sterile TE to final concentration with10ng/µl.
2. Prepare reaction mixture as given below:Components---------- Volume (µl)PCR buffer (pH 8.3) ----------5dNTP (2.5mM each) ---------4Taq polymerase (1U/µl) -----1Template DNA (10ng/µl) ----1RAPD-Primer (10µM) -------810% DMSO ------------------5DDW ------------------------26Final Volume-----------------503. Operate the thermal cycler program as given below:Initial denaturation------- 94ºC-------5mi -----Single stepDenaturation -------------94ºC -------1minAnnealing ----------------36ºC -------1min ----30 CyclesExtension ----------------72ºC -------2minFinal extension -----------72ºC -------5min ----Single step4. Perform agarose gel electrophoresis as described in Expt. 6.
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Labels: A DEMONSTRATIVE MANUAL FOR BASIC MOLECULAR BIOLOGY PRACTICAL
Sunday, July 8, 2007

OPTIMIZATION OF RAPD-PCR FOR GENETIC FINGERPRINTING OF BACILLUS THURINGIENSIS
Gyan Sundar Sahukhal1, Upendra Thapa Shrestha1, Kiran Babu Tiwari1, Binod Lekhak2, Anjana Singh2, Viswanath Prasad Agrawal*(1) Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Universal Science College, Maitidevi, Kathmandu, Nepal;(2) Central Department of Microbiology, Tribhuvan University, Kirtipur, Nepal* Corresponding Address: Dr. Vishwanath P. Agrawal, Professor of Biochemistry, Universal Science College, Pokhara University, Kathmandu. Email: vpa@wlink.com.npABSTRACT
A random amplified polymorphic DNA fingerprinting assay has been optimized that discriminate different B. thuringiensis isolates. Random amplification of polymorphic DNA (RAPD) is proving to be a useful technique used for screening diversity, particularly at intraspecific levels, including many population studies. Relationships between species' may be determined by comparing their unique fingerprint information, which are expected to be identical among related species. The technique can be troublesome and time consuming to establish due the essentially empirical approach to optimization. By standardization of certain parameters and use of a commercially available PCR buffer, a particularly promising primer was identified and a RAPD condition for a highly discriminatory and reproducible characterization of B. thuringiensis isolates was achieved. In addition, a technique to obtain reproducible RAPD fingerprints of B. thuringiensis without the need to purify genomic DNA described.


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*)
(1) Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Universal Science College, Maitidevi, Kathmandu, Nepal;
(2) Central Department of Microbiology, Tribhuvan University, Kirtipur, Nepal
*Address for Correspondence: Prof. Dr. Vishwanath Prasad Agrawal, RLABB, Tel: +977-1-4442775, E-mail: vpa@wlink.com.np
Journal of Food Science and Technology Nepal. 2: 128-131
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 themethod 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
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 determinationFive 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 bioassayThe 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 involvingreversible 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 ELISADouble 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.
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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.
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Labels: Delta-endotoxin Immuno Cross-reactivity of Bacillus thuringiensis Isolates Collected from Khumbu Base camp of Mount Everest Region

Strong mosquitocidal Bacillus thuringiensis from Mt. Everest
Upendra Thapa Shreshtha1, Kiran Babu Tiwari1, Anjana Singh2, Vishwanath Prasad Agrawal1*(1) Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Universal Science College, Maitidevi, Kathmandu, Nepal;(2) Central Department of Microbiology, Tribhuvan University, Kirtipur, Nepal*Address for Correspondence: Prof. Dr. 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 146), 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 altitude where mosquitoes are not expected, may be novel. The S6 isolate 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).
Fig 1: % Mortality of larvae
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

Cloning of cry3 fragment in Escherichia coli

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: +977-1-2110043ABSTRACT
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.
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.
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.
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Labels: Cloning of cry3 fragment in Escherichia coli
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KHUMBU REGION-THE RESEARCH AREA OF RLABB







Starting of my research with Bacillus thuringiensis endotoxin
Delta Endotoxin-The Insecticidal Crystal Protein

3D STRUCTURE OF DELTA ENDOTOXIN
Delta Endotoxin - The Crystal Protein