IGEM:Cambridge/2008/Turing Pattern Formation/Experiments/Bacillus subtilis transfomation

From OpenWetWare
Jump to navigationJump to search

Transformation of Bacillus Subtilis

Our aim

We want to transform Bacillus Subtilis with two kinds of vectors: episomal vectors and integration vector. We would like to obtain good efficiency, no contamination and some precise tests confirm our transformations and to be able to standardize our protocol

Material

  • 3 different strains:
    • IA1
    • IA751
    • IA771 which contains erm insertion at the amyE locus, so transformants at the amyE locus can be screened for erythromycin resistance
  • 15 different vectors which have been gel-tested (correct size achieved with double digests)
    • 10 integration vectors (7 validated)
    • 5 shuttle vectors (3 validated)


Media Preparation

  • 10X Medium A base:

- Yeast extract 10g

- Casamino acids 2g

- Distilled water to 900mL

- Autoclave, then add :

- 50% glucose, filter sterilized 100mL

  • 10X Bacillus salts:

- (NH4)2SO4 20g

- Anhydrous K2HPO4 139.7g

- KH2PO4 60g

- Tri-sodium citrate 10g

- MgSO4•7H2O 2g

- SDW to 1000mL

  • Medium A

- Sterile water 81mL

- 10X Medium A base 10mL

- 10X Bacillus salts 9mL

- L-Tryptophan (11mg/mL) 0.1mL

  • Medium B

- Medium A 10mL

- 50mM CaCl2•2H2O 0.1mL

- 250nM MgCl2•6H2O 0.1mL


Important:

- Autoclave Medium A base before adding glucose, and autoclave Bacillus salts

- Store aliquots of 10X Medium A base 10mL and 10X Bacillus salts 9mL and keep them in the fridge, never use them twice to avoid contamination

Protocols

  • Making Bacillus competent

1- Grow one blank plate of Bacillus Subtilis (or several if you want to transform different strains) for 20 hours at 37ºC (plate been kept on the bench for several days would be better)

2- Inoculate about 12mL of medium with several colonies. Mix the contents of the tube. Check with OD650. Start OD should be between 0.1 and 0.2. Be careful to pipette 0.8mL of this mixture into the cuvette to measure and dispose of it after measurement to avoid contamination in the main mixture.

3- Incubate at 37ºC with vigorous shaking. Read the OD650 every 20min (never keep the solution you used for measuring!)

4- Plot log(OD650) in function of time. After a brief lag, you should observe a exponential increase. After awhile, it will leave the exponential growth; the moment at which it leaves the exponential path is denoted as t0 (3 on the graph). It should take about 100min and the OD should be between 0.35 and 0.55.

Retrieved from "https://openwetware.org/mediawiki/index.php?title=IGEM:Cambridge/2008/Turing_Pattern_Formation/Experiments/Bacillus_subtilis_transfomation&oldid=238959"