User:James Chappell/ Integration
- pMUTIN4 = RBS http://www.bgsc.org/NewProducts/pMUTIN4.pdf
- 1A806 Bacillus subtilis subsp. subtilis Cm pyrD::cat RM35
- 1A807 Bacillus subtilis subsp. subtilis Km pyrD::kan RM301
- 1A808 Bacillus subtilis subsp. subtilis Cm gltA::cat RM36
- 1A809 Bacillus subtilis subsp. subtilis Km gltA::kan RM37
- 1A810 Bacillus subtilis subsp. subtilis Cm sacA::cat RM58
- 1A811 Bacillus subtilis subsp. subtilis Km sacA::kan RM182
- B. subtilis strains were grown in LB broth (1% tryptone, 0.5% yeast extract and 0.5% NaCI)
at 37~ with vigorous aeration to an OD600 of 1.5-2.0.
- Cells were harvested and washed three times in sterile cold de-ionised water by centrifugation at 3000 g for 10 minutes and finally resuspended in 30% polyethyleneglycol (PEG) 6000 (BDH, biochemistry grade) to 1% of the original culture volume.
- The ceils were then dispensed into 100 μl aliquots, rapidly frozen and stored at -70~ Where cells
were harvested and suspended in either 15% glycerol or a range of PEG concentrations, they were used immediately.
- Electroporation was performed using a Bio-Rad Gene Pulser and Pulse Controller as
described by Dower et aL (1988). Cells to be transformed were first thawed on ice, 500 ng DNA in 10μl of water added, gently mixed and transferred to a pre-chilled 0.2 cm gap eleetroporation cuvette.
- The cells were then immediately pulsed, diluted in 2 mlSOC broth (2% tryptone, 0.5% yeast extract,
10 mM NaCI, 2.5 mM KC1, 10 mM MgC12, 10 mM MgSO4, 20 mM glucose) and transferred to 15 ml Falcon centrifuge tubes. Cells were incubated at 37~ with gentle shaking for 90 minutes to allow expression of the antibiotic resistance. For chloramphenicol and erythromycin resistant plasmids, 0.2/~g/ml of the appropriate antibiotic was added to the transformed cells in SOC after 60 rains incubation to induce resistance. Cells were then suitably diluted and plated out onto nutrient agar (Oxoid) alone or containing the appropriate antibiotic at 5 /~g/ml for chloramphenicol and erythromycin and 25 ~g/ml for tetracycline.
- Electroporation was performed using an Eppendorf Electroporator 2510 with 2mm band gap cuvettes
- One microliter DNA was combined with 50µl cells, and a pulse of 2300 volts applied
- Cells recovered for three hours in SOC medium before plating on NB agar supplemented with the appropriate antibiotics.
To isolate DNA, single colonies of transformed B. subtilis were innoculated into 5ml Nutrient Broth and incubated in a round-bottom culture tubes with shaking at 30°C for 18 or 22 hours. From each culture, cells were centrifuged as 4000rpm at 4°C for 10 minutes. Cell pellets were thoroughly resuspended in 250µl Cell Resuspension Solution (Promega Corporation, Madison, WI) and transferred to a sterile 1.5ml microcentrifuge tube. DNA was isolated from one-half of the samples using the standard Wizard® Plus SV Miniprep DNA Purification System protocol described in Technical Bulletin #TB225 (Promega Corporation, Madison, WI). One-half of the samples were treated with 100µl of lysozyme (10mg/ml) for 15 minutes at 30°C prior to adding the Cell Lysis Solution from the DNA purification system and proceeding with the protocol described in Technical Bulletin #TB225. Absorption of purified DNA samples was measured at A260, and the DNA concentration for each sample was calculated (Table 1).
I can't say for sure for B. subtilis, but with S. aureus we do the following: Grow up cells to OD 0.6 Wash 4x successively in 0.5, 0.25, 0.125, and 0.0625 times the original culture volume of ice-cold, sterile, 0.5M sucrose at one of these steps, incubate 2-4 hrs on ice. We don't know why, but I have a feeling it might be to elute all the salt from the cell wall (?) resuspend cells in 10% glycerol, 4ml per liter original culture volume snap freeze in LN2 in 50ul aliquots