User:Uriel E Barboza Perez/Notebook/Transformation of Bacillus thuringiensis 4Q7 With cry1Ac gene

Transformation of an Acrystalliferous strain Bacillus thuringiensis 4Q7 with the cry1Ac gene

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

People who worked on this project:

• Carolina Guizar Bravo
• Mariana Cazares
• Brenda Briones
• Uriel E Barboza Perez

Project Description/Abstract

The main goal of this project is only for academic purposes only. Our goal was only to design and to do a genetic Engineering project as a final examination of our Genetic Engineering class at ITESM campus qro.

Background

B.thuringiensis insecticides are popular with organic farmers because they are considered "natural insecticides" and differ from most conventional chemical insecticides as they are toxic to only a small range of related insects and are not harmful to mammals, birds or fish.The insecticidal crystals of B. thuringiensis are formed by Cry (crystal) and cytolytic (Cyt) proteins, most of them formed during the sporulation state, and constitute 20-30% of the cell dry weight. These crystals contain millions of copies of Cry or Cyt proteins that interact by disulfide bridges or by ionic interaction to form visible crystals under phase contrast microscopy . Currently there are reported aprox. 728 and 37 different types of Cry and Cyt proteins, respectively . The Cry proteins inside the crystals are called protoxins, because they are the nontoxic protein. However, when a susceptible insect consumes the crystal, protoxins are released and transformed by proteolytic enzymes to the toxic protein called delta-endotoxin.

Objective

The goal of our project was to transform an Acrystalliferous strain of Bacillus thuringiensis Serovar Israelensis Strain 4Q7 with a shuttle vector pht3101 containing the Cry1Ac gene and ecoli top 10 with the same vector , in order to compare the crystal production under phase contrast microscopy.

This plasmid and strain was kindly given to us by Barboza Lab

Pht3101 Vector

Materials for BT transformation

Reagents	Amount
E. coli Top10	---
LB agar plates	2
LB	300ml
CaCl2 0.1M + 15% glycerol solution	100ml
CaCl2 0.01M	100ml
Agarose gel 0.7%	300ml
Distilled water
TAE1x	700ml
50ml tube	1
sterile tubes 1.5ml	---
Microcentrifugue tubes	---
Ice	---
micropipete 200ul	1

Materials for miniprep

Reagents	Amount
BT4Q7 on a nutrient agar plate	1
BHI medium

Protocols

[show][hide]

Making LB Agar plates

• Preparation

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

• Antibiotics

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 [math]\displaystyle{ CaCl2 }[/math] 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.

Notes:

• 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

• 1. Thaw competent cells on ice for 10 minutes.
• 2. Add 1-3 μL prepped plasmid DNA to each 100 μL aliquot and incubate on ice for at least 30 minutes.
• 3. Transfer to 42°C for 2 minutes.
• 4. Put on ice for 5 minutes.
• 5. Add 900 μL of LB + 20 mM glucose or SOC and incubate at 37 °C with shaking or rotation for at least one hour.
• 6. Plate on selective media and incubate overnight at 37 °C.

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 Electrophoresis

• Gel preparation:

1. 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.
2. Heat the mixture in the microwave until the powder has completely dissolved stirring the contents every so often.
3. Transfer the solution into a disposable container.
4. Gel stains should be added when the agarose becomes cool enough to touch.(For SYBR Safe gel, add 5μl to 50ml TAE buffer)

• Electrophoresis setting:

1. 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.
2. Pipette a small amount of the tepid gel mixture around the edges of the taped regions to seal the chamber.
3. Add remaining gel solution to the chamber, and wait to set. The comb can then be removed from the chamber.
4. 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).
5. 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.
6. 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

Lab Notebook/log

October

[06/10/14]

• Sterilization of material
• Step 1 of competent cells

[07/10/14]

• Step 2 of competent cells
• Step 1.a of Miniprep Invitrogen Protocol

[08/10/14]

• Step 3 to 9 of competent cells

[09/10/14]

• Run electrophoresis for the plasmid extraction aliquots

[10/10/14]

• 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.

November

[09/11/14]

• 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

Reagents	Amount
LB broth	5ml
Miniprep Invitrogen kit®	1

Reagents	Amount
Enzyme	1μl
dH20	9μl
Dna	8μl
Buffer	2μl

[11/11/14]

We prepared 100 ml of the following mediums and we then autoclaved them.

Brain heart infusion

Brain heart infusion Agar

Nutritive Agar

Once the mediums were autoclaved , Bt 4Q7 was cultured in 2 nutritive agar plates. They were incubated and left overnight at 37 C.

[12/11/14]

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.

[13/11/14]

We didn't have the material ready so we inoculated in Bt 4Q7 in new BHI liquid medium

[14/11/14]

Bt 4Q7 was transformed with the vector PHT101- cry1Ac with electroporation.

[15/11/14]

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.

1) B.Thuringiensis 4Q7

2) B.Thuringiensis 4Q7-PHTCry1AC

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.

1) Ecoli Top 10

2) E.coli Top10-pHT3101 cry1Ac

[16/11/14]

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.

Protein separation from Bacillus thuringiensis (Bt). Lane M, Protein marker (Invitrogen); lane 1 Bt 4Q7; lane 2, Bt 4Q7/pHT3101-cry1Ac. The arrow indicates the position of the Cry1Ac protein.

Conclusions

we are cool .

Notes

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

Recently Edited Notebook Pages

• 2017-09-27 00:31:14 User:Uriel E Barboza Perez/Notebook/Transformation of Bacillus thuringiensis 4Q7 With cry1Ac gene/Entry Base