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From 2012.igem.org

Contents 1 Prepare Bs competent cells from strains of interest 2 Bs strains growth rate data 3 Resuspend lyophilized primers for cross-over PCR 4 Make O/N cultures (2x) of pBR322-transformed XL1-Blue competent cells

Prepare Bs competent cells from strains of interest

• Prepare fresh SpC medium (4x 20 mL in 125-mL E. flasks) by adding the following to 40 mL 5X Spizizen salts solution in a 50-mL conical tube:
  • 400 μL 50% (w/v) glucose
  • 800 μL 10% (w/v) Bacto-yeast extract
  • 1 mL 1% (w/v) casein hydrolysate
  • To one 50-mL tube, add 400 μL of 2 mg/mL L-Trp for the two auxotrophic strains (168 and PERM739)
• Take 150 μL each of SpC and SpC-Trp medium for blanks in a 96-well plate
• Divide into 4x 125-mL E. flasks and pre-warm to 34^o C
• Scrape cell growth off each plate from the night before and inoculate the respective flasks
• Incubate at 34^o C with vigorous aeration (240rpm) while periodically taking 150 μL samples for OD₆₀₀ readings to obtain an approximate growth curve for each strain (below)

• While waiting for cells to grow, prepare fresh SpII medium (4x 200 mL in 1-L E. flasks) by adding the following to 400 mL 5X Spizizen salts solution
  • 4 mL 50% glucose
  • 4 mL 10% Bacto-yeast extract
  • 4 mL 1% casein hydrolysate
  • 2 mL 0.1 M CaCl₂
  • To one 400 mL solution, add 4 mL 2 mg/mL L-Trp for the two auxotrophic strains (168 and PERM739)
• Divide into 4x 1L E. flasks and pre-warm to 34^o C
• When cell growth rate departs from exponential (i.e. no significant change in OD over 20-30 min), inoculate 200 mL of pre-warmed SpII medium with 2 mL of stationary-phase cultures and incubate at 34^o C with slower aeration (160rpm)
  • Note: When I checked on the cultures at the 70-75 min mark, the incubator was at the right temperature but not shaking. This may have greatly reduced the growth achieved.
• After 90 minutes of incubation, pellet the cells by centrifugation (4,000 g, 12 min) at RT
  • Note: The protocol has the centrifugation at 8,000 g for 5 min, but the centrifuge used had a max rpm of 4,000. This may have reduced the percentage of cells pelleted
  • For this step, ~50 mL of each culture were put in 50-mL conical tubes and centrifuged serially to obtain one pellet per strain
• Decant supernatant into sterile container and save
• Gently resuspend cell pellet in 18 mL of saved supernatant and add 2 mL sterile glycerol, mix gently by pipetting up and down
• Aliquot competent cell cultures (~0.5 mL) in sterile tubes
  • Purple (36) - PERM739
  • White (36) - 168
  • Green (36) - PY79
  • Yellow (32) - 1A833
• Store at -80^o C for several years

Bs strains growth rate data

Growth rate data was obtained by collecting samples for OD₆₀₀ readings every hour for the first 3 hours, then every half hour until stationary phase was reached at 5 hours. Table 1 shows the raw absorbance values at each time point before normalization against the two blanks of SpC (for prototrophic PY79 and 1A833) and Spc-Trp (for auxotrophic 168 and PERM739). Well A1 contained 150 μL of SpC medium (OD₆₀₀ = 0.086), and well A2 had 150 μL of SpC-Trp medium (OD₆₀₀ = 0.087). Figure 1 shows the normalized data plotted...

Table 1. Raw (not normalized) absorbance values

Time (hrs.)	168	PERM739	PY79	1A833
0a	0.219	0.392	0.257	0.398
1	0.275	0.459	0.316	0.492
2	0.444	0.570	0.492	0.655
3	0.732	0.739	0.768	0.848
3.5	0.827	0.776	0.841	0.874
4	0.875	0.829	0.915	0.894
4.5	0.919	0.841	0.921	0.924
5	1.022	0.917	0.966	0.932

Notes: ^a The discrepancies of the readings at the start of incubation (time of 0 hrs.) are from a lack of fine control in the amount of cells initially scraped off each plate. Perhaps interestingly, however, the two ancestral strains (168 and PY79) were less dense than their ΔmutSL derivatives.

Resuspend lyophilized primers for cross-over PCR

• Spin down tubes
• Add the volume specified in the table of TE Buffer to the primer tubes

Name	Volume (uL)
pBf	190.55
pBr	136.9
p2f_2	205.7
p2r_2	196.25
p3f_2	207.85
p4r_2	185.65

• Vortex all tubes briefly
• Spin all tubes down
• Incubate on bench for 10 minutes
• Make 10 μM working solutions by adding 38 μL RNase/DNase-free water to 2 μL stock solutions
• Store stock solutions at -20 ^oC, and working solutions at 4^o C

Make O/N cultures (2x) of pBR322-transformed XL1-Blue competent cells

• Inoculate a single colony from the fresh plate made yesterday into 2 mL LB-Amp broth
• Incubate at 34^o C overnight