IGEM:IMPERIAL/2009/Assays Protocols/Cloning strategy M2

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(New page: Image:cloningstrategym2.jpg Cloning strategy for colonic acid 1. Ligate cI responsive promoter e.g. Bba_R0051 (P3) to RcsB (G4) 2. Test for colonic acid production 3. Remove term...)
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Cloning strategy for colonic acid
Cloning strategy for colonic acid
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1. Ligate cI responsive promoter e.g. Bba_R0051 (P3) to RcsB (G4)
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1. Ligate cI responsive promoter e.g. Bba_R0051 (P3) to RcsB (G4)  
2. Test for colonic acid production
2. Test for colonic acid production
 +
*'''[[User:James Chappell|James Chappell]] 09:16, 29 July 2009 (EDT)''':Normally for testing we use well characterised inducible promoters, not sure the cI responsive promoter is the best opition for this. how about using a lac promoter for testing of RscB.
3. Remove terminators from the P3+RcsB construct
3. Remove terminators from the P3+RcsB construct
 +
*'''[[User:James Chappell|James Chappell]] 09:16, 29 July 2009 (EDT)''':As we said yesterday once you have built you biobrick devices you cannot delete any of the parts it contains only add to them either in front or behind.
4. Ligate B3023 construct. Test for increased colonic acid production
4. Ligate B3023 construct. Test for increased colonic acid production
 +
*'''[[User:James Chappell|James Chappell]] 09:16, 29 July 2009 (EDT)''':Whats B3023? wouldnt you want to decouple its affect on colonic acid production from RscB?
5. Remove terminators from the P3+RcsB+B3023 construct
5. Remove terminators from the P3+RcsB+B3023 construct

Revision as of 09:16, 29 July 2009

Image:cloningstrategym2.jpg

Cloning strategy for colonic acid

1. Ligate cI responsive promoter e.g. Bba_R0051 (P3) to RcsB (G4)

2. Test for colonic acid production

  • James Chappell 09:16, 29 July 2009 (EDT):Normally for testing we use well characterised inducible promoters, not sure the cI responsive promoter is the best opition for this. how about using a lac promoter for testing of RscB.

3. Remove terminators from the P3+RcsB construct

  • James Chappell 09:16, 29 July 2009 (EDT):As we said yesterday once you have built you biobrick devices you cannot delete any of the parts it contains only add to them either in front or behind.

4. Ligate B3023 construct. Test for increased colonic acid production

  • James Chappell 09:16, 29 July 2009 (EDT):Whats B3023? wouldnt you want to decouple its affect on colonic acid production from RscB?

5. Remove terminators from the P3+RcsB+B3023 construct

6. Ligate Waal ligase and perform the colonic acid assay


Cloning strategy for trehalose production

1. Ligate P3 to OtsA.

2. Test for trehalose production.

3. Remove terminators from P3+OtsA construct

4. Ligate OtsB to P3+OtsA construct

5. Test for enhanced trehalose production


Trehalose and colonic acid

1. Ligate +RcsB+B3023+Waal Ligase construct with terminators, and P3+OtsA+OtsB construct with terminators.

2. Test for both colonic acid and trehalose production

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