IGEM:IMPERIAL/2009/Assays Protocols/Cloning stratedy M2
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 terminators from the P3+RcsB construct
4. Ligate B3023 construct. Test for increased colonic acid production
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