Paris/ConstructionProcess

From 2007.igem.org

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Our aim is to have the following construction inserted into the genome:
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Our aim is to have the following construction inserted into the genome: (est-ce que tu pourrais faire le schéma nicolas ?)
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Strong promoter - lox71 - RBS-GFP-Ter - FtsK (with its own promoter) - terminator - lox66 - dapA
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Strong promoter - lox71 - RBS-GFP-terminator - FtsK (with its own promoter) - terminator - lox66 - dapA  
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giving after recombination:
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Strong promoter - lox scar - dapA
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 +
There are two different ways to do this:
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 +
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.
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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.

Revision as of 15:20, 19 October 2007


Our aim is to have the following construction inserted into the genome: (est-ce que tu pourrais faire le schéma nicolas ?)

Strong promoter - lox71 - RBS-GFP-terminator - FtsK (with its own promoter) - terminator - lox66 - dapA

giving after recombination:

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


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