Streptococcus Transformation

Generic Protocol

• Preheat 3 ml of media 'A' using a heat block set at 37 degrees.
• Inoculate fresh colonies (1-2 days old -- use a significant number) in the 3ml of media 'A'.
• Preheat a 96-well plate with 270ul media 'B' to 37 degrees using the 37 degree incubator with ambient CO2.
• Occasionally, check for growth to appropriate Absorbance. When the cells have grown enough, add the following to the 96-well plate:
  • Add 2ul CSP-1 peptide (more than 100 ng/ml eventual final concentration. In aliquots in the -80 freezer; 20ul aliquots of 100uM. Talk to Eric if you are not working with a D39 derivative).
  • Add DNA to 1 ug/ml final concentration — so 300ng.
  • Add 30ul of grown cells (a 1:10 dilution).
  • Pipet up and down to mix; incubate for 60 minutes in the 37 degrees 5% CO2 incubator.
• Warm up 2.7ml TSB in a test tube for each transformation using the hot block.
• At the end of the 60 minute incubation, add all 300ul of the transformation to each TSB test tube.
• Incubate for 60 minutes (or longer) in the 37 degrees 5% CO2 incubator.
• Either:

1. 1. Pre-warm blood plates with appropriate antibiotics OR on a TSA plate with appropriate antibiotics. Label plates.
   2. Add 6-7 glass beads
   3. Plate 500ul of sample AND 25ul added catalase (if using a TSA plate), put onto the plate the same time as the bacteria
   4. Use beads to distribute.
   5. Allow plates to completely dry in hood.

• • Or,

1. 1. During this second 60 minute incubation, change the hot block to 42 degrees and put in 2 empty test tubes per transformation.
   2. Melt and pipet 3.5ml TSB soft agar into each test tube.
   3. Warm up TSB plates with appropriate antibiotics by placing them in either 37 degree incubators. Move to the hood and label plates.
   4. 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.
   5. 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.
   6. Repeat the above step with a second plate, so that all 3ml of the transformation is used.
   7. 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.
   8. Wait 3-5 minutes for all plates to solidify

• Incubate for 1-2 days in 37 degree, 5% CO2 incubator

Specifics, by transformation media

Media 'A' (Growth Media)	Media 'B' (Transformation Media)	Grow to Absorbance
C+Y pH 7.4	C+Y pH 7.4	0.52 Absorbance
CTM pH 6.8	CTM pH 7.8	0.39 Absorbance
BHI pH 7.4	430ul culture + 50ul 100mM NaOH + 5ul 20% BSA + 2ul 100mM CaCl2	0.1 Absorbance
Columbia pH 7.4	Columbia pH 6.6	0.05 Absorbance. With our Spectrometer, impossible. Use 0.13 Absorbance; dilute 4-fold with pre-warmed Columbia pH 7.4; wait 30 minutes and then use.

Recipes

C+Y Recipe

Complete Transformation Medium (CTM)

• 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 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.