B. subtilis
PROTOCOL DUMP PLEASE FORMAT
*Reference: Bron, S. (1990) Plasmids. In Molecular Biological Methods forBacillus. Harwood, C.R., and Cutting, S.M. (eds). Chichester, UK: John Wiley & Sons, pp. 75–174.`
Solutions to prepare BEFORE the Day of Transformation
1) 10x PC solution (Autoclave to sterilise)
Total Volume (make up to this volume) 200 mL
K2HPO4(anhydrous) 21.4 g
KH2PO4(anhydrous) 12 g
Na3citrate.2H2O 1.7 g
[Sigma-Aldrich#S4641]
2) 20% (w/v) Glucose Solution (Filter Sterilise)
3) L-tryptophan (0.2%) 2 mg mL-1 (Filter Sterilise) total 25 mL
[Sigma-Aldrich#T0254] *B. subtilis168 is a tryptophan mutant; hence, it is critical to add this aa.
Note: Tryptophan is light sensitive. Wrap a VWR tu`be in tin foil and store in fridge.
4) DL aspartic acid potassium salt 50 mg mL-1 (Filter Sterilise) total 25 mL
[Sigma-Aldrich#11240]
5) MgSO41M (Autoclave to sterilise) total 25 mL
6) 20% Casamino acids (Casein Hydrolysate/CAA) (w/v) (Autoclaved to Sterilise) total 25 mL
[Sigma-Aldrich#22090] *When taken out of the autoclave, the Casein Hydrolysate will have mostly dissolved in water. However, on returning to room temperature it will crash out of solution. On the day of the experiment, incubate at 60 °C for a few hours to partially re-dissolve. Then, vortex again and pipette using a wide-opening pipette tip (LTS tips 1000 µL). Alternatively, microwave in 20 second intervals, stirring after. Around 3 20-second intervals will be needed until the solution turns a dark red. It won’t be fully dissolved; but that’s ok.
Putting the 25 mL solutions in VWR 50 mL containers in 4C is good idea!
To prepare on the Day of Transformation
7) Ammonium iron (III) citrate 2.2 mg mL-1 (Filter Sterilise) total 1 mL
[Sigma-Aldrich#F5879]
The above solutions are mixed to make the MD Medium
MD Medium
Final Volume
50 mL
30 mL
10 mL
10x PC
5 mL
3 mL
1 mL
20% glucose (w/v)
5 mL
3 mL
1 mL
L-tryp (0.2%) 2mg mL-1
1.25 mL
750 uL
250 uL
Ammonium iron(III) citrate 2.2mg mL-1
250 uL
150 uL
50 uL
DL aspartic acid 50 mg mL-1
2.5 mL
1.5 mL
500 uL
MgSO41M
150 uL
90 uL
30 uL
Transformation Protocol
Empty flask of water and add 10 mL MD medium + 50 µL 20% casamino acids/casein hydrolysate
Keep remaining MD medium at 37 °C.
Inoculate with small loopful of freshly-streaked strain or ~20 µL an overnight culture (final solution must not be turbid)
Shake at 37 °C (vigorous-top rack) until OD600 reaches 1-1.5 max(Dilute 100µL in 1 mL to measure OD, ).This takes approximately 4-5 hr.
Add an equal volume (10 mL) of warm MD medium lacking casamino acids. Shake at 37 °C for 1 hr. Cells will now be competent. Add 800 µL of competent cells to pre-warmed 10 mL plastic falcon tube for each transformation. Add DNA to final concentration of 1 µg mL-1. (Control with no DNA)
Shake at 37 °C for 20 min and then add 25µL of 20% casamino acids to each transformation tube (or LB medium).
Shake at 37 °C for 1 hr longer or 80 min for erythromycin expression.
Plate on selective antibiotic plates (if needed) with 1% starch from potato and incubate at 30 °C. eg. 3 plates with 300 µL each. If no antibiotic resistance, dilute 1000x and plate 1µL.
White/cream coloured colonies should appear on the plates in 16-20 hr. * UnlikeE. coli,B. subtiliscolonies are not circular; they have irregular/ undulate margins and tend to spread out and merge with each other rather quickly.See image.
Screening Method
To screen the colonies for disruption of theamyElocus (hence, loss of starch hydrolyzing ability), add 4-5 mL of Gram’s Iodine solution [VWR# 101411-034] to the plate to cover the colonies (do this in the fume hooda), nd incubate at room temperature for 1 min. The starch-agar background will turn black. Colonies withNO clear zone/ halo around themhave disruptedamyEand these are the ones that should be picked for further analysis. See comparison atsource.
You’re looking for Amylase (-), which indicates removal ofamyE.
amylase +
amylase -
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