Streptococcus Transformation

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Reagents Needed

  • CTM (Complete Transformation Medium) pH 6.8
  • CTM pH 7.8
  • CSP-1 peptide (in the -80 freezer; 20ul aliquots of 100uM) or CSP-2 peptide (which we do not have). Talk to Eric if you are not working with a D39 derivative.

Complete Transformation Medium

  • 3g Tryptic Soy Broth
  • 0.1g yeast extract
  • Fill up to 100ml MilliQ water and autoclave
  • Add to a final concentration filter sterilized 1mM CaCl2 (found on chemical shelf), filter sterilized 0.2% BSA (Bovine Serum Albumin), and filter sterilized 1X trace mineral solution (found on chemical shelf)

Competence Protocol

  1. Freshly grow up single colonies on a blood TSA plate of the strain to be transformed.
  2. Select one colony and grow in 3ml CTM pH 6.8 until OD 0.3, which is 0.39 Absorbance
  3. Preheat a microcentrifuge tube of 270ul CTM pH 7.8 to 37 degrees using the hot block.
  4. Add CSP-1 peptide to this tube to at least 100 ng/ml eventual final concentration. We use 2ul of the CSP-1 aliquot, which brings the concentration to 228ng/ml.
  5. Add DNA to 1 ug/ml final concentration — so 300ng. If this is too much DNA, it might work with half of the amount — 150ng of DNA.
  6. Add 30ul of grown cells (a 1:10 dilution).
  7. Vortex
  8. Incubate at 37 degrees for 60 minutes using the hot block.
  9. During this time, warm up 2.7ml CTM pH 6.8 in a test tube for each transformation using the hot block.
  10. At the end of the 60 minute incubation, add all 300ul of the transformation to each test tube. Be sure to label which test tube is which.
  11. Incubate for 60 minutes in the 37 degree incubator.
  12. During this second 60 minute incubation, change the hot block to 42 degrees and put in 2 empty test tubes per transformation.
  13. Melt and pipet 3.5ml soft agar (either TSB or HTY) into each test tube.
  14. Warm up TSB plates with appropriate antibiotics by placing them in either 37 degree incubators. Move to the hood and label plates.
  15. After the second 60 minute incubation, add 10ul of catalase and 10ul of TCC to each soft agar tube. There's no need to add antibiotics to the soft agar.
  16. Add 2 x 750ul of the transformation to a soft agar tube (using a micropipettor, not a serological pipet), immediately vortex for 3-5 seconds and immediately pour onto a TSB plate. The agar will solidify quickly, so do this step one soft agar tube at a time.
  17. Repeat the above step, so that all 3ml of the transformation is used. For (-), no DNA controls, feel free to only plate a total of 1.5ml onto a single TSB plate. Repeat for all transformations, one soft-agar tube at a time.
  18. Wait 3-5 minutes for all plates to solidify
  19. Incubate the plate overnight in the 37 degrees C 5% CO2 incubator.

Competence Protocol with CRISPR

  • As above, but add the editing construct at a final concentration of 0.7 - 2.5 ug/ml (210ng - 750ng total DNA)
  • Incubate at 37 degrees using the hot block for 20 minutes.
  • Add the CRISPR targeting construct at a final concentration of 0.7 - 2.5 ug/ml (mirroring the first set of DNA), and vortex.
  • Incubate at 37 degrees using the hot block for 40 minutes.