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

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m (Test for colonic acid)
m (Test for colonic acid)
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Legend: ABR = Antibiotic resistance gene. We don't actually need it!!!
Legend: ABR = Antibiotic resistance gene. We don't actually need it!!!
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*'''[[User:James Chappell|James Chappell]] 15:14, 29 July 2009 (EDT)''':be careful about copying last years cloning strategy too much, we also do not need the 5' and 3' biobricks. Also please say what G2, G3 etc is some where.
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*'''[[User:James Chappell|James Chappell]] 15:14, 29 July 2009 (EDT)''':'''Overall Feedback''' be careful about copying last years cloning strategy too much, we also do not need the 5' and 3' biobricks. Also please say what G2, G3 etc is some where. Also think you need lot more combinations of inducible promoter and a gene (see comment below for more info).
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<u>Phase 1: Testing of promoters</u>
<u>Phase 1: Testing of promoters</u>

Revision as of 15:19, 29 July 2009

Image:cloningstrategym2.jpg


  • James Chappell 09:18, 29 July 2009 (EDT):General feedback - Have a look at last years cloning strategy, it can really help to plan wet lab CLICK HERE TO SEE PNG. Also would be good to put names on biobrick pictures.

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


Test for colonic acid

Legend: ABR = Antibiotic resistance gene. We don't actually need it!!!

  • James Chappell 15:14, 29 July 2009 (EDT):Overall Feedback be careful about copying last years cloning strategy too much, we also do not need the 5' and 3' biobricks. Also please say what G2, G3 etc is some where. Also think you need lot more combinations of inducible promoter and a gene (see comment below for more info).

Phase 1: Testing of promoters

1. Testing of P1:how varying arabinose causes variation in GFP

Image:testp1.jpg

2. Testing of P2: inhibition of P2 by G2

Image:testp2.jpg

3. Testing of P3: inhibition of P3 by G3

Image:testp3.jpg

Phase 2: Testing of genes

1. Testing to see if RcsB works:

Colonic acid assay will be performed. Production of colonic acid is a proof of a positive test.

Image:m2genet1.jpg

Perhaps we could do this to simplify matters???

Image:m2genet1m.jpg

2. Testing to see if the RcsB+B3023 works:

  • James Chappell 15:17, 29 July 2009 (EDT):It would be lot easier to test if the colonic acid production works by putting the B3023 under the direct control of an inducible promoter. For me this would be the first test before I get into a lengthy cloning process to produce this larger construct.

Colonic acid assay will be performed. Increased production of colonic acid is a proof of a positive test.

Image:m2genet2.jpg

3. Testing to see if RcsB+B3023+Waal ligase works.

A different form of colonic acid will be produced. Try washing colonic acid off. Colonic acid still remaining is a proof of a positive test.

Image:m2genet3.JPG

Test 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


Test for both trehalose and colonic acid production

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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