Question

1. Vibrio cholerae is a naturally competent bacterium. You have 2 strains of V. cholerae: a ciprofloxacin-resistant strain and a ciprofloxacin-sensitive strain. Create a procedure to transform the cipro-sensitive strain with the cipro-resistant DNA over 4 lab periods. Make sure you include reagents, materials, details, and controls.
   Lab week 1:

Lab week 2:

Lab week 3:

Answer 1

Week 1.

• Grow resistant bacteria. Using standard LB liquid medium, add the adequate ammount of antibiotic to it (this medium must contain antibiotic to guarantee that only resistant bacteria will grow). Be careful to keep sterile conditions, use a burner and, if possible, a laminar flow bell. Distrubute the medium in test tubes, and inoculate using the tip of an sterile toothpick. Grow overnight at ~37°C.
• Extract plasmidic DNA. Using an standard MiniPrep procedure, lysate the bacteria, and isolate plasmidic DNA. Preferably use the entire content of each test tube, this way you will have the most DNA possible. You should pellet the bacteria in a centrifuge, get rid of the medium, fill the tube again with the bacterial suspension and pellet again. Lysate using lysis buffer, centrigufe to get rid of cellular debris, and purify the plasmid using chloroform (I could go into way more detail here, but this would be a 10-page answer. A standard MiniPrep protocol can be easily found online. If your teacher is one fo those guys that, for some reason, wants every single detail, you can copy/paste the protocol. He/She probably will not even read it).
• Run plasmid gel. Prepare a 1% garose gel, using TAE buffer and agarose. When it is still hot and liquid, add ethidium bromide (or you can add it later, depends on your lab). Mix ~6uL of electrophoresis buffer and ~1uL extracted DNA. Load the gel's wells and run at ~120 volts for ~20 minutes. You should see 3 bands in the gel. If you do, you are doing great. If not, well, verification will confirm.
• Verify by PCR. To make sure the DNA you extracted is the correct one, perform a PCR using primers for the antibiotic resistance gene AND another PCR for a different part of the plasmid. This will make sure that your extract has the plasmid and the resistance gene. You will need:
  • PCR buffer
  • Taq Polymerase
  • MgCl2
  • Forward and Reverse primers
  • Distilled or milliQ water
  • dNTPs
  • your DNA.
  • For the procedure, mix everything but the DNA in a 1.5mL microtube. Distribute the mix to 20uL nanotubes and THEN add the DNA. The volume of DNA you add varies depending on the quantitation, but since this PCR is for verification only, you can just go for it with 1uL of DNA (exactitude is not vial at this stage). Set the thermocycler for 25 cycles and run the reaction. (Again, a lot more detail could be included here, but this would be unbearable. If necessary, find the protocol online and copy/paste to satisfy your teacher as needed). After PCR, run a gel as described above. You should see a band of the total size of your plasmid and a band the size of your resistance gene. If you do not get those bands, it is time to trace back and start over.

This should keep you busy the first week.

Week 2.

• Quatitate plasmid. This step depends on you lab equipment. But generally, it is some spectrometric method. Load ~1uL of extracted DNA in the plate/cell, measure absorbance and calculate concentration accordingly (there are different formulas for this, depends on the equipment). Some equipments directly give you the concentration. If the concentration is not enough, amplify with PCR as described above.
• Adjust concentration. Once you got your concentrations, adjust them to ~100 or ~50 ng/uL using sterile water.
• Transfrom replication verctor (E. coli) to clone plasmid. In order to use the plasmid to transform the sensible batecria, it needs to be cloned. For this, E. coli is used. First, thaw your cloning strain. The transformation can be performed in a variety of ways, in this case let's use electroporation. Put a swab of almost-frozen bacteria in the electrporation cell. Add ~10uL of plasmidic DNA (concentration 50ng/uL). Put the electroporation cell in the electroporation machine and press the button to electroporate. This takes abut 3 seconds (yes, it is that simple). Take the electroporated cells and plate them in LB agar + antibiotic. This will kill cells that did not transform and leave you the colonies that DID transform. You can keep the E. coli transformants in the fridge at 4°C for several weeks, long enough for you to finish the transformtion.

Week 3.

• Extract plasmid from E. coli. Using the same MiniPrep protocol as before, extract plasmid from E. coli. Run a gel to verify that you have the three bands.
• Verify by PCR. Using the same PCR protocol as before, verify that your extract contains the parts you need (the resistance gene).

Week 4.

• Grow sensitive V. cholerae. Using liquid LB medium, and following the same steps as in Week 1, grow V. cholerae overnight. Quantitate ammount of cells in the suspension using the spectrophotometer.
• Transform using extracted plasmid. Use the same transformation protocol via electroporation as described above. Plate electroporated bacteria in LB agar + antibiotic, to kill non-transformants and keep transfrmants.
• Isolate transformants and pass to liquid antibiotic medium. Once the colonies have grown on the agar medium with antibiotic, use an sterile toothpick to inoculate test tubes filled with liquid LB + antibiotic. Repeat this a couple times to maintain the resistance. If you do not inoculate the resistant bacteria in antibiotic medium a couple times, the bacteria will "spit out" the plasmid. You need to keep the selective pressure for a couple generations so that the resistance plasmid stays in.
• Freeze. Freeze your new resistant bacteria by pelleting, adding 50% glycerol and dropping them in liquid nitrogen. Put away in a Revco at -80°C for later use.