Streptococcus Transformation

Transformation Protocol with CTM media (edited 25 March 2026)

1. Preheat 3 ml of CTM pH 6.8 using a heat block set at 37 degrees. Create a tube with 9ml CTM pH 6.8 that you will use to blank the spectrophotometer.
   • • • One option is to aliquot this out into sterile test tubes the night before; place in the 37 degree incubator to pre-warm these tubes + media overnight.
2. Using a cotton swap, inoculate fresh colonies in the 3ml of CTM pH 6.8. Aim to add enough colonies that the CTM is slightly cloudy (but not over an Ab of 0.1).
   • • • Colonies should not be more than 1 day old
       • The more colonies you inoculate, the faster the media will be ready.
3. Using a mini vortex inside of the biological hood, vortex cultures to break up colonies using a mini and incubate in the 37 degree / 5% CO2 incubator.
4. Preheat a 96-well plate with 270ul CTM pH 7.8 [note different pH] using the 37 degree incubator with ambient CO2. You will need one well for each strain+DNA combination, plus one for a negative, cell-only control.
5. Grow cells to an Absorbance of >0.15, but not over 0.4, using the spectrophotometer. Be sure to blank the spectrophotometer with your tube with just CTM. If the culture is over 0.6, you will need to start the culture over from colonies on a plate. Cells between Ab 0.4 and 0.6 can be diluted down, using the prewarmed CTM from the blank tube with just CTM.
6. When the cells have grown enough, dilute to ~0.18 Absorbance.
7. Add the following to the 96-well plate:
   1. 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).
   2. Add DNA to 1 ug/ml final concentration — so 300ng.
   3. If working with a Gibson assembly, add 10uL of the Gibson Assembly, straight from making it, in place of the DNA.
   4. Add 30ul of grown cells (a 1:10 dilution).
   5. Pipet up and down to mix.
8. Incubate for 15-30 minutes in the 37 degrees 5% CO2 incubator. Longer is better, until 30 minutes; then, success decreases.
9. During this incubation, warm up 2.5ml TSB to 37 degrees in a test tube for each transformation using the hot block. Label these test tubes.
   • • • This will allow for cultures to acclimate to TSA when plated.
10. At the end of the transformation incubation, add all 300ul of the transformation to each TSB test tube.
11. Incubate for 30-60 minutes in the 37 degrees 5% CO2 incubator.
    • • • Expression of antibiotic resistance proteins/markers occurs during this step; do not go over 60 minutes, as cells will start to divide.
12. During this second incubation, change the hot block to 42 degrees and put in 1 empty test tube per transformation.
13. Melt and pipet 3.5ml TSB soft agar into each test tube.
14. Warm up TSB plates with appropriate antibiotics by placing them in either 37 degree incubators. They need to be at room temperature or higher. Move to the hood and label plates.
15. After the second incubation, add 5ul of catalase and 5ul of TTC to each soft agar tube. There's no need to add antibiotics to the soft agar.
16. Add 1000ul 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 warmed, labeled, TSB plate that has the appropriate antibiotic. The agar will solidify quickly, so do this step one soft agar tube at a time.
17. Repeat for all transformations, one soft-agar tube at a time.
18. Wait 5-10 minutes for all plates to solidify
19. Incubate overnight in 37 degree, 5% CO2 incubator

• • Visible colonies will form after 12 hours. Plates can grow for up to 48 hours. If these strains contain a New Sweet Janus cassette, using colonies that appear 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.

Complete Transformation Medium (CTM)

1. 7.5g Tryptic Soy Broth
2. 0.25g yeast extract
3. Fill up to 250ml MilliQ water
4. Separate into 200ml and 50ml in graduated cylinders
5. Change pH of the 200ml to pH 6.8
6. Change pH of the 50ml to pH 7.8
7. Put into labeled bottles; add on the label "◻ Additives".
8. Autoclave the same day
9. 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)
10. Once these extra bits are added, check off the label "☑️ Additives"

Older notes

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C+Y Recipe

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.

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.