Paris/ConstructionProcess
From 2007.igem.org
Our aim is to have the following construction inserted into the genome: Strong promoter - lox71 - RBS-GFP-terminator - FtsK (with its own promoter) - terminator - lox66 - dapA
giving after recombination / differentiation:
Strong promoter - lox scar - dapA
There are two different ways to do this:
1. You can try to clone this whole construction on a plasmid, then insert it in the chromosome and finally, you would need to delete the FtsK gene.
2. You can also clone separately the upstream part (Promoter – lox71 - RBS-GFP-terminator) and the downstream part (terminator - lox66 – dapA). Then you only need to insert these two construct upstream and downstream of the natural FtsK gene.
After a failed attempt to use the first method, we opted for the second one.
In fact, we first attempted to use the first method with FtsA + FtsZ instead of FtsK. Mutant of those genes basically give the same phenotype as FstK mutant, that is to say filamentous cells. But FtsA + FtsZ proved to be very difficult to clone. The expression balance of those genes seems indeed to be of a crucial importance for the cell survival, making any cloning attempt a nightmear if the insert is only slightly expressed.
After a month and a half of failed clonings, we moved on to the second method and chose FtsK instead of FtsA+FtsZ. FtsK is indeed an isolated gene, which is not the case of FtsA and FtsZ present in a large operon. This is a very important point for the second method. We are going to frame FtsK with our up/downstream constructs. Doing so, we do not want to disturb the expression of potentially important surrounding genes.
Contents |
Construct : Forward construction
Construct : Backward construct
Intermediate construct
Backward construct with DapA E.Coli
Backward construct with DapA Subtilis
Construct : recombinaison rate measurement
