Streptococcus Transformation

Transformation Protocol with CTM media

• Preheat 3 ml of CTM pH 6.8 using a heat block set at 37 degrees.
• Inoculate fresh colonies in the 3ml of CTM pH 6.8. Vortex ultures to break up colonies.
  • Colonies should not be more than 1-2 days old
  • The more colonies you inoculate, the faster the media will be ready for step 2.
• Preheat a 96-well plate with 270ul CTM pH 7.8 using the 37 degree incubator with ambient CO2.
• Grow cells to an Absorbance of >0.15. Make sure to vortex before checking Absorbance. When the cells have grown enough, dilute to 0.05 Absorbance.
• 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.
  • If working with Janus Cassette, add 15uL.
  • 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.
  • Must be TSB. Allows for cultures to acclimate to TSA when plated.
• At the end of the 60 minute incubation, add all 300ul of the transformation to each TSB test tube.
• Incubate for 60 minutes in the 37 degrees 5% CO2 incubator.
  • Also works if only incubated for 30 minutes. Expression of antibiotic resistance proteins/markers occurs during this step.

• 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
      1. Add beads first then plate cultures so that cultures don’t splash everywhere if you throw beads onto a plate with liquid.
   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.
   6. Incubate plates in the 37°C, 5% CO2 incubator.
      1. Visible colonies will form after 12 hours. Plates can grow for up to 48 hours. If these strains contain a Janus cassette, using these cultures earlier is better. This will lower odds of mutations occurring in the rpsL or sacB genes, which are responsible for streptomycin and sucrose resistance/sensitivity.

• • 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

For transformation not using CTM media, use the following media combinations below.

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.