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Inducing Bacillus subtilis with Subtilin
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Riachalder (Talk | contribs) (New page: * Make ON cultures of the transformed ''B. subtilis'' and ''B. subtilis'' ATCC6633 wild type * Add 50µl ''B. subtilis'' ON culture to 2mL LB. * Incubate for 1 hour at 37°C. (''This is th...) |
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* Make ON cultures of the transformed ''B. subtilis'' and ''B. subtilis'' ATCC6633 wild type | * Make ON cultures of the transformed ''B. subtilis'' and ''B. subtilis'' ATCC6633 wild type | ||
* Add 50µl ''B. subtilis'' ON culture to 2mL LB. | * Add 50µl ''B. subtilis'' ON culture to 2mL LB. | ||
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* Incubate for 3 hours at 30°C. (''This is the optimum temperature for the reporter genes used (''gfp'' and ''mCherry'') as these are isolated from the cnidarians ''Aequorea victoria'' and ''Discosoma striata respectively. This enhances expression of the reporter genes.'') | * Incubate for 3 hours at 30°C. (''This is the optimum temperature for the reporter genes used (''gfp'' and ''mCherry'') as these are isolated from the cnidarians ''Aequorea victoria'' and ''Discosoma striata respectively. This enhances expression of the reporter genes.'') | ||
* Divide the 2mL diluted culture into 2 screw-cap culture tubes and add subtilin to one (the other will be a control. To obtain subtilin, centrifuge the wild type ON culture and collect the supernatent. This should contain sectreted subtilin protein. Add this in the desired concentration (e.g for 1% induction, use 10µl supernatent in 1mL diluted culture.) | * Divide the 2mL diluted culture into 2 screw-cap culture tubes and add subtilin to one (the other will be a control. To obtain subtilin, centrifuge the wild type ON culture and collect the supernatent. This should contain sectreted subtilin protein. Add this in the desired concentration (e.g for 1% induction, use 10µl supernatent in 1mL diluted culture.) | ||
| - | * Incubate for | + | * Incubate for 2 hours to allow induction of the insert and reporter gene. |
| + | * Proceed to [[Preparing Bacillus subtilis for Microscopy]] | ||
Latest revision as of 16:10, 28 October 2008
Newcastle University
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Home >> Wet Lab >> Protocols >> Inducing Bacillus subtilis with Subtilin
- Make ON cultures of the transformed B. subtilis and B. subtilis ATCC6633 wild type
- Add 50µl B. subtilis ON culture to 2mL LB.
- Incubate for 1 hour at 37°C. (This is the optimum temperature for B. subtilis
- Incubate for 3 hours at 30°C. (This is the optimum temperature for the reporter genes used (gfp and mCherry) as these are isolated from the cnidarians Aequorea victoria and Discosoma striata respectively. This enhances expression of the reporter genes.)
- Divide the 2mL diluted culture into 2 screw-cap culture tubes and add subtilin to one (the other will be a control. To obtain subtilin, centrifuge the wild type ON culture and collect the supernatent. This should contain sectreted subtilin protein. Add this in the desired concentration (e.g for 1% induction, use 10µl supernatent in 1mL diluted culture.)
- Incubate for 2 hours to allow induction of the insert and reporter gene.
- Proceed to Preparing Bacillus subtilis for Microscopy
