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 cultures 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:
  • Pre-warm blood plates with appropriate antibiotics OR on a TSA plate with appropriate antibiotics. Label plates.
  • Add 6-7 glass beads
    • Add beads first then plate cultures so that cultures don’t splash everywhere if you throw beads onto a plate with liquid.
  • Plate 500ul of sample AND 25ul added catalase (if using a TSA plate), put onto the plate the same time as the bacteria
  • Use beads to distribute.
  • Allow plates to completely dry in hood.
  • Incubate plates in the 37°C, 5% CO2 incubator.
    • 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,
  • During this second 60 minute incubation, change the hot block to 42 degrees and put in 2 empty test tubes per transformation.
  • Melt and pipet 3.5ml TSB soft agar into each test tube.
  • Warm up TSB plates with appropriate antibiotics by placing them in either 37 degree incubators. Move to the hood and label plates.
  • 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.
  • 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.
  • Repeat the above step with a second plate, 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.
  • 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.