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

Phase 1: Testing of promoters

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

2. Testing of P2: inhibition of P2 by G2

3. Testing of P3: inhibition of P3 by G3

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.

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

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

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

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