User:Uriel E Barboza Perez/Notebook/Transformation of Bacillus thuringiensis 4Q7 With cry1Ac gene
|E. coli Top10||---|
|LB agar plates||2|
|CaCl2 0.1M + 15% glycerol solution||100ml|
|Agarose gel 0.7%||300ml|
|sterile tubes 1.5ml||---|
For Bacillus Thuringiensis Transformation
|Bt 4Q7||50ul /aliquot---|
|BHI Liquid Medium||10ml|
|40% PEG 6000 (w/v)||1ml|
|0.2cm electroporation cuvette||300ml|
|Culture tube (17 x 100)||1|
|Sterile tubes 1.5ml||5|
|Shuttle vector pht3101 containing the Cry1Ac gene||5ul /aliquot ---|
|Miniprep Invitrogen kit®||1|
Making LB Agar plates
Prepare liquid broth (LB) from liquid broth capsules and distilled water.
- Making LB Agar (15%)
- 1. In a 1 litre bottle, add 3.75g of Agar powder to 250ml LB
- 2. Autoclave to sterilize
- 3. These can be stored and heated up to make plates when needed
Different antibiotics will have different optimum concentrations
- Chloramphenicol (25mg/ml)
Add 250mg to Chloramphnicol to 10ml ethanol (100%). Use 1ml for 1 litre of LB Agar to make final concentration 25ug/ml plates.
- Making LB Agar Plates
- 1. Cool down/heat up LB agar to approx. 55 degrees Celsius
- 2. In a flow hood, add 250ul antibiotics to 250ml LB agar; mix
- 3. Pour approx. 25ml into each plate
- 4. Wait approx. 30 mins for plates to set
- 5. Store in 4 degrees Celsius fridge for future use
Preparation of competent cells
- 1. Subculture an E.coli overnight culture 1:100 in LB (e.g. 500 μL overnight in 50 ml LB in a 250 ml flask). Incubate at 37°C with shaking to an OD600 of 0.375.
- Culture growth beyond OD 0.4 decreases transformation efficiency.
- 2. Aliquot 20 ml of the culture into chilled 50 ml tubes. Leave the tubes on ice for 5-10 minutes.
- Keep cells cold for all subsequent steps.
- 3. Centrifuge cells for 7 minutes at 1600 g, 4°C. Allow centrifuge to decelerate without brake.
- 4. Discard supernatant and resuspend each pellet in 4 ml ice cold CaCl2 solution.
- 5. Centrifuge cells for 5 minutes at 1100 g, 4°C.
- 6. Discard supernatant and resuspend each pellet in 4 ml ice cold CaCl2 solution. Keep on ice for 30 minutes.
- 7. Centrifuge cells for 5 minutes at 1100 g, 4°C.
- 8. Discard supernatant and resuspend each pellet in 800 μL ice cold CaCl2 solution.
It is important to resuspend this pellet well.
- 9. Aliquot 100 μL of this suspension into microcentrifuge tubes. Freeze in liquid nitrogen and store at -80°C.
Miniprep Invitrogen Protocol
- 1.a. Inoculate the target bacteria in 1-5 mL medium broth (LB, YT o Terrific Broth) for 24 hours at 37°C. If the plasmid carries any marker, for example antibiotic resistance, add into the medium the marker substance.
• Preheat an aliquot of TE Buffer (TE) to 65–70°C for eluting DNA. Heating is optional for eluting 1– 30 kb plasmid DNA but is recommended for eluting DNA >30 kb.
• Caution: Buffers contain hazardous reagents. Use caution when handling buffers.
- 1. Harvest. Centrifuge 1–5 mL of the overnight LB-culture. (Use 1–2 × 109 E. coli cells for each sample.) Remove all medium.
- 2. Resuspend. Add 250 μL Resuspension Buffer (R3) with RNase A to the cell pellet and resuspend the pellet until it is homogeneous.
- 3. Lyse. Add 250 μL Lysis Buffer (L7). Mix gently by inverting the capped tube until the mixture is homogeneous. Do not vortex. Incubate the tube at room temperature for 5 minutes.
- 4. Precipitate. Add 350 μL Precipitation Buffer (N4). Mix immediately by inverting the tube, or for large pellets, vigorously shaking the tube, until the mixture is homogeneous. Do not vortex. Centrifuge the lysate at >12,000 × g for 10 minutes.
- 5. Bind. Attach the spin column with the supernatant from step 4 to a luer extension of the vacuum manifold. Apply vacuum. After all of the supernatant has passed through the column, turn off the vacuum.
- 6. Optional Wash. (Recommended for endA+ strains). Add 500 μL Wash Buffer (W10) with ethanol to the column. Incubate the column for 1 minute at room temperature. Apply vacuum. After all of the liquid has passed through the column, turn off the vacuum.
- 7. Wash. Add 700 μL Wash Buffer (W9) with ethanol to the column. Apply vacuum. After the liquid has passed through the column, turn off the vacuum.
- 8. Remove ethanol. Place the column into a 2-mL wash tube. Centrifuge the column at 12,000 × g for 1 minute. Discard the wash tube and flow-through.
- 9. Elute. Place the spin column in a clean 1.5-mL recovery tube. Add 75 μL of preheated TE Buffer (TE) to the center of the column. Incubate the column for 1 minute at room temperature.
- 10. Recover. Centrifuge the column at 12,000 × g for 2 minutes. The recovery tube contains the purified plasmid DNA. Discard the column. Store plasmid DNA at 4°C (short-term) or store the DNA in aliquots at −20°C (long-term).
Heat Shock Transformation
Artificial transformation is a process whereby E.coli are made competent and take up DNA from their surroundings. The main steps are chilling the cells to 0oC with CaCl2 solution, adding the DNA and heat shocking the bacteria for a short period of time, allowing the cells to recover at 37oC and then plate them.
One hypothesis for artificial transformation is that the divalent cation provided by the chilled CaCl2 solution which is used in creating competent cells promotes an interaction between DNA and lipopolysaccharides which are also negatively charge. Lipopolysaccharides occur in higher densities near the parts of the outer membrane of Gram negative bacteria that are in close association with the inner membrane (zones of adhesion).
It is believed that prior to incubation DNA may interact with lipopolysaccharides and then cross the 'least barrier path' at the zones of adhesion described above.
Ensure that you have enough sterilized liquid broth (or another medium such as SOC) and agar plates containing the correct antibiotic. Before you begin have a waterbath set to 42oC and an incubator at 37oC.
Take care not to disturb the competent E.coli: do not vortex them or pipette them up and down.
- Thaw competent E.coli cells ( 50 μl in an eppendorf tube, grown to an OD600 of 0.2 - 0.5) on ice. Let them sit on ice for at least 10 minutes.
- Add 1-3 μl of plasmid and mix gently by stirring with the pipette tip. Use more plasmid solution if it is very weak. We have had success with about 50 ng of plasmid DNA.
- Hold on ice for 0.5 hours
- Heat shock the cells with the DNA at 42ºC for 1 minute.
- Add 250 μl of SOC medium.
At this stage it is important to maintain sterility to avoid contaminating the liquid broth or the plates. Do not allow the pipette tip to touch anything before pipetting the liquid broth. Leaving the liquid broth bottle on the bench overnight should reveal any contamination in the morning.
- Incubate at 37ºC for 1 hour.
- Plate 10μl on agar containing an antibiotic (the antibiotic for which the plasmid confers resistance) and also plate 100μl on another identical plate. This allows for possible overcrowding so that single colonies can be selected.
- Incubate overnight at 37ºC.
Always keep agar plates upside down so that drips of condensation and falling debris does not contaminate them.
BT transformation procedure
- 1) Inoculate the freshly-grown single colony of BT 4Q7 (on a nutrient agar plate) into 10 (or more 50-100 ml depend how much you cells you want) of BHI (Brain Heart Infusion) medium.
- 2) Let the culture grow for 12 hr at 30 ºC with shaking (250 rpm).
- 3) Incubate the culture on ice for 10 min
- 4) Harvest: 1,000 x g for 10 min at 4 ºC
- 5) Washing: 1,000 x g for 10 min (x 3 times) at 4 ºC using 10 ml of ice-cold sterilized ddw
- 6) Resuspend pellet in 1 ml of ice-cold 40% PEG 6000 (w/v)
- 7) Incubate the cells on ice for 10 min
- 8) Make 300 μl aliquot using 500 μl tubes
- 9) Add 2 to 5 μg of plasmid DNA into an aliquot and mix
- 10) Transfer the mixture into a 0.2 cm cuvette
- 11) Single pulse: 2.3 kV, 480 Ohm, 25 μF (¡ these are different that those used to transform E. coli!).
- 12) Add 3 ml of BHI medium into the mixture immediately
- 13) Transfer the whole thing into a 17 x 100 mm culture tube
- 14) Incubate the tube for an hour at 37 ºC with gentle shaking
15) Plate it out using BHI plates containing appropriate antibiotics 16) Usually colonies will appear in less than 24 hr.
PEG: Polietilenglicol 6000
- Gel preparation:
- For 1% agarose gel (say 200ml), add 2g of agarose powder to 200 ml of 1x TAE buffer (obtained by diluting 10xTAE stock buffer)
- Note: The shorter the DNA strand lengths, the more concentrated the gel will be.
- Use 75-100ml of buffer for preparing one gel.
- Heat the mixture in the microwave until the powder has completely dissolved stirring the contents every so often.
- Transfer the solution into a disposable container.
- Gel stains should be added when the agarose becomes cool enough to touch.(For SYBR Safe gel, add 5μl to 50ml TAE buffer)
- For 1% agarose gel (say 200ml), add 2g of agarose powder to 200 ml of 1x TAE buffer (obtained by diluting 10xTAE stock buffer)
- Electrophoresis setting:
- Ensure electrophoresis chamber is clean and dry, tape the sides (with Autoclave tape, NOT standard masking tape) to make watertight. Slot in the desired comb.
- Pipette a small amount of the tepid gel mixture around the edges of the taped regions to seal the chamber.
- Add remaining gel solution to the chamber, and wait to set. The comb can then be removed from the chamber.
- Fill the electrophoresis apparatus half-full with 1x TAE buffer solution (for good electrical contact) and place the set gel in the buffer. Ensure that there are no air bubbles (particularly in the wells created by the comb).
- Add the ladder solution to the first well, and the DNA samples to subsequent wells. A loading dye may be added to the mixtures to aid visualisation when loading into wells.
- Connect the electrodes to the apparatus (the right way round!). Set DC voltage at 80V (with current at approximately 3 mA) and run for 30-60 minutes (or until the DNA has separated sufficiently).
- Tips for a Successful Gel:
- Add buffer, not water, when making the gel
- Seal the gel mould using autoclave tape (not masking tape) and with hot agarose
- After boiling buffer and agarose, let it cool before pouring into mould to prevent leakage
- Use running buffer to lubricate removal of mould else risk breaking the wells
- High salt is bad so dilute sample after enzymatic reactions
- Use full volume of well
- Check DNA is running towards the positive/cathode/red pole
- Check that your voltage and current are appropriate; running gel too fast will distort the bands
- Use fresh buffer for each gel, as a pH gradient will build up during each run
Transformation of the Bacteria
E. coli was cultured in LB agar and incubated at 37ºC over night. Subsequently Electrocompetent E. coli cells were prepared and then transformed via electroporation with pHT3101-Cry1AC in order to obtain a higher concentration of pHT3101-Cry1AC. The transformed E. coli were cultured in LB agar containing Ampicillin at 100 mg/ml and incubated at 37ºC.
Bt 4Q7 was cultured in two Nutrient Agar plates. They were incubated and left overnight at 37 ºC. 2 single colonies were picked and inoculated in Brain-Heart Infusion (BHI) liquid medium. They were left at 37ºC overnight and transformed with the vector PHT101- cry1Ac via electroporation.
Verification of plasmid insertion
After 24 hours the plasmid from the E. coli TOP10 was isolated using the Biorad Miniprep Kit. An Agarose gel Electrophoresis was done to verify the size of the construct. The Agarose was 0.8% and it was run at 90V for aprox 1 hr.
For the Bt 4Q7, the plasmid extraction was done using the PureLink® Quick Plasmid Miniprep Kit from Invitrogen. After plasmid isolation an agarose gel was performed to check the size of our plamids. This gel was run with a positive control of the PHT101- cry1Ac vector that was originally given to us in order to verify our plasmid with the Positive control, isolated plasmids extracted from recombinant E.coli pHT3101-cry1Ac and Bt 4Q7/pHT3101-cry1Ac.
A polyacrylamide gel was also done in order to separate the proteins from Bt4Q7/pHT3101-cry1Ac in order to verify if the protein was being produced by the strain.
Polyacrylamide gel was performed at 10% (1.9 mL of sterile distilled water, 1.7 mL acrylamide 30%, 1.3 mL of Tris-HCl pH 8.8 1 M, 50 mL of sodium dodecyl sulfate (SDS) at 10%, 50 mL of ammonium persulfate (PSA) and 6 mL of Temed) under denaturing conditions in a vertical electrophoresis chamber Mini-PROTEAN Tetra System (Bio-Rad).
After the separating gel solidified, the stacking gel was performed , (1.4 mL of sterile distilled water, 330 mL of 30% acrylamide, 250 ul of Tris-HCl pH 6.8 1 M, 20 mL of 10% SDS, 25 mL of PSA to 5 mL of Temed). Running buffer pH 8.3 Tris-glycine was added to the camera. From a sporulated culture, 1. 2 mL were taken, centrifuged and the pellet was washed twice with water to remove residues. 150 uL of 5x Laemmli buffer supplemented with β-mercaptoethanol was added to the pellet and it was boiled for 10 min and centrifuged at 14,000 rpm for 2 min. 15 uL of each sample were taken and placed in the wells respectively. To determine the molecular weight of the marker proteins the BenchMark Prestained Protein (Invitrogen) was used. The gel was run at 100 V for 2:30 hours. After finishing the gel run, the gel was stained with Coomassie blue (2.5 g Coomassie Brilliant Blue G, 450 mL methanol, 90 mL of acetic acid and 460 mL of distilled water) for 2 hours with stirring and it was destained with a solution of 20% acetic acid (v / v) (100 mL of acetic acid, 400 mL of distilled water). Finally the gel was analyzed in the Quantity One program fotodocumentador "Gel Doc" (BioRad).
Phase Contrast Microscopy
E. coli, and 4Q7were cultivated in LB at 37°C or 28°C (200 rpm), respectively. Samples were taken at different times and monitored by phase contrast microscopy. Data was obtained using an Axio Imager A.1 Zeiss microscope with the filter set at 09, an excitation of 450–490 nm, and an emission of 515 nm.
- Sterilization of material
- Step 1 of competent cells
- Step 2 of competent cells
- Step 1.a of Miniprep Invitrogen Protocol
- Step 3 to 9 of competent cells
- Run electrophoresis for the plasmid extraction aliquots
- Transform E.coli by heat shock (protocol)
A plasmid extraction was done using the PureLink® Quick Plasmid Miniprep Kit from Invitrogen
2 aliquots were obtained and marked with numbers 1 and 2.
- Agarose gel Electrophoresis of isolated plasmid
An Agarose gel Electrophoresis was done to verify the size of our isolated plasmid( Note** our plasmid was not linearized)
The Agarose was 0.8% and it was runned at 90V for aprox 1 hr.
The results were the following:
The aliquots 1 and 2 had the same size indicating they contained the same plasmid.However the bands were above the actual size of our plasmid. This could be due to the closed form of the vector so in order to verify its actual size we must linearize the vector. To linearize the plasmid an enzyme digestion was done with Sph1 and left overnight at 37 C
A 20 μl reaction was done
We prepared 100 ml of the following mediums and we then autoclaved them.
Brain heart infusion
Brain heart infusion Agar
Once the mediums were autoclaved , Bt 4Q7 was cultured in 2 nutritive agar plates. They were incubated and left overnight at 37 C.
The bacteria grew on the plates as expected:
Bt4Q7 on Nutritive Agar
2 single colonies were picked and inoculated in two falcon tubes containing BHI liquid medium. They were left at 37 C overnight.
We didn't have the material ready so we inoculated in Bt 4Q7 in new BHI liquid medium
Bt 4Q7 was transformed with the vector PHT101- cry1Ac with electroporation.
A plasmid extraction was done using the PureLink® Quick Plasmid Miniprep Kit from Invitrogen
After plasmid isolation an agarose gel was performed to check our plamids.
This gel was runned with a positive control of the PHT101- cry1Ac vector that was originally given to us in order to verify our plasmid with the Positive control
Phase contrast microscopy was done in order to verify the formation and the expression of the cry1Ac gene in Bt 4Q7. The results were positive!!! :) and the next image illustrates it.
In these images we can clearly observe that the expression of the cry1Ac gene led to effective crystal formation in image2. In the image 1 there are no crystals present and we can only observe the natural spores of Bacillus thuringiensis
One of the main objectives of this project was also to compare the expression of cry genes in ecoli. We knew that ecoli wasnt able to express crystal forms inside the cell.
This was confirmed with phase contrast microscopy. In image 1 we have ecoli top 10 and in image 2 , ecoli top10 with our vector containing the cry1Ac gene.
There was no significant difference between the normal ecoli and the recombinant strain of ecoli, confirming our hypothesis.
A polyacrylamide gel was done in order to separate the proteins from Bt4Q7/pHT3101-cry1Ac. We used the following protocol:
Analysis of the gel was done and the results of our gel where the following.
Results And Discussion
E.coli expresses but doesn’t produce the Cry1Ac crystals
When E. coli was transformed with the vector containing the cry1ac gene ,i.e pHT3101-cry1Ac (Figure 1A). In order to verify the expression of the cry1AC gene in recombinant E. coli- pHT3101-cry1Ac an agarose gel electrophoresis was performed. The cry1ac gene was observed in the gel indicating that the recombinant E.coli in fact contained the cry1Ac protein (Figure 1B). Wiltype E.coli and recombinant E.coli and were observed under phase contrast microscopy and the results showed no presence of crystals in neither of the strains.
PHT3101-cry1Ac accumulates in the form of crystals in acrystalliferous Bacillus thuringiensis 4Q7
When pHT3101-cry1Ac was introduced in 4Q7, the recombinant bacterium produced crystals in the cytoplasm, readily detected by phase contrast microscopy. (Figure 2A). Recombinant 4Q7 was compared with the wiltype form of Bacillus 4Q7 and the results in fact showed the presence of crystals in the recombinant strain. To further our studies, an agarose gel and a protein gel was performed to verify the gene insert in the recombinant Bacillus 4Q7. The agarose gel was perfomed by running the vector in both, circular an linear form. To linearize the plasmid an enzyme digestion was done with SalI and the results showed in figure 2B confirm the presence of the gene. To verify the presence of the cry protein the SDS PAGE was preformed only in Bacillus thuringiensis 4Q7 and, Bt 4Q7/pHT3101-cry1Ac.The results in supplementary figure 2C show a band corresponding to the cry1AC in the recombinant 4Q7 that prove the presence of the protein in Bt 4Q7/pHT3101-cry1Ac
Bacillus thuringiensis as a factory to produce proteins that E. coli can’t produce due to its less complex synthetizing machinery.
We have so far demonstrated that E.coli is able to replicate the cry1Ac gene but isn’t able to produce crystals , and also that the cry1Ac accumulates in the form of crystals in the acrystalliferous strain of Bacillus thuringiensis 4Q7.
In order to further our studies, and to confirm the presence of pHT3101-cry1Ac in both recombinant E.coli and Bacillus thuringiensis. ,an agarose gel electrophoresis was run with the original vector pHT3101-cry1Ac , isolated plasmids extracted from recombinant E.coli pHT3101-cry1Ac and Bt 4Q7/pHT3101-cry1Ac . The results showed the presence of the same gene in both of the recombinant strains of E.coli and bacillus 4Q7 indicating that our recombinant bacteria in fact contained the same plasmid.
As expected, Bt 4Q7 showed production of the crystals under phase contrast microscopy and In Figure 2A Panel 2 we can see the crystal protein that is composed of Cry Proteins along with typical spores found in Bacillus thuringiensis. Compared to Bt 4Q7, transformed E. coli Top 10 didn’t show production of the crystals as seen in Figure 1C panel 2, confirming our hypothesis that Bacillus thuringiensis Serovar Israelensis Strain 4Q7 is able to produce a well-shaped crystal due to its more complex machinery and that it can be used as a factory to produce proteins that E. coli can’t produce.
We would like to thank M.S. Karen Stephania González-Ponce for providing us the pHT3101-Cry1AC and Dr. Eleazar Barboza Corona for the Bacillus thuringiensis Serovar Israelensis Strain 4Q7 and for letting us use the Quantity One program fotodocumentador "Gel Doc" (BioRad) and the Axio Imager A.1 Zeiss microscope to analize our results. Special thanks to Dr. Estibaliz Sansinenea for providing us with the article “Genetic manipulation in Bacillus thuringiensis for strain improvement”. And last but not least, we thank Dr. Digpal Singh Gour for his teachings and the support during our project.
Barboza Corona J.E. & Ibarra J.E. Proteínas Insecticidas de Bacillus thuringiensis. Boletín de Educación Bioquímica-ISSN: 1665-1995 05/1998; 17(1):3-10.
Barboza Pérez U.E & Barboza Corona J.E. A bacterial factory that produces insecticidal, antimicrobial and chitinolytic proteins with biotechnological uses. Insights in Biotechnology 05/2014. Vol 7. 5-7 Bravo (1), A., Soberón, M. (n.d.) Bacillus thuringiensis y sus toxinas insecticidas. [Online book chapter] Retrieved from: http://www.biblioweb.tic.unam.mx/libros/microbios/Cap12/
Bravo (2), A., Soberón, M. (2007, november 14) Las toxinas Cry de Bacillus thuringiensis: modo de acción y consecuencias de su aplicación. Retrieved from: http://www.ibt.unam.mx/computo/pdfs/libro_25_aniv/capitulo_27.pdf
Jung YC1, Mizuki E, Akao T, Côté JC. (2007, July) Isolation and characterization of a novel Bacillus thuringiensis strain expressing a novel crystal protein with cytocidal activity against human cancer cells. Retrieved from: http://www.ncbi.nlm.nih.gov/pubmed/17584453 Sansinenea E ,Vázquez C, Ortiz A. (2010). Genetic manipulation in Bacillus thuringiensis for strain improvement. Biotechnology Letters. DOI: 10.1007/s10529-010-0338-
This work was done during the semester( august 2014-Dic 2014) at the genetic engineering lab of our campus as part of our biotech courses. If you want more info on our protocols or if you wish to have this plasmid to do this experiment for your classes feel free to contact me at firstname.lastname@example.org