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-	Our aim is to have the following construction inserted into the genome:	+	The aim of the Molecular biology work was to construct the backbone for SMB genomic cassette:
		+
	[[Image:Paris_FinalConstruct.JPG]]		[[Image:Paris_FinalConstruct.JPG]]
-	giving after recombination / differentiation:	+	In order to construct this genomic cassette, we have considered 2 solutions:
		+
		+
		+	* 1)Either the construct is cloned on a plasmid. Then inserted in the genome strain of a dapA-, ftsK-(ts)  E.coli strain. This was the initial strategy we adopted then subsequently abandoned (see here why LIEN).
		+	* 2)Or the cassette is sequentially assembled, in the genome, around the essential gene selected (ftsK in our case).
		+
		+	We have chosen the second strategy. The different steps of this process are as follow:
		+
		+	2 plasmid born constructs were generated. These are termed the UP “upstream construct” & the DP “downstream construct”:
-	Strong promoter - lox scar - dapA	+	Upstream construct:
-	There are two different ways to do this:	+	[[Image:Paris_UpstreamConstruct.JPG]]
-	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.	+	Downstream construct :
-	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.	+	[[Image:Paris_DownstreamConstruct.JPG]]
-	After a failed attempt to use the first method, we opted for the second one.	+	As their names indicate, these constructs are to be inserted upstream and downstream of ftsK in the genome. We chose to insert these constructs using a strategy described by Wanner et al.
-	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.	+	We have yet to insert the constructs in the genome. This will be done sequentially:
		+	[[Image:Paris_UpstreamInsertion.JPG]]
-	After a month and a half of failed clonings, we moved on to the second method and chose FtsK instead of FtsA+FtsZ.	+	[[Image:Paris_FlpRec.JPG]]
-	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.	+
		+	[[Image:Paris_DownstreamInsertion.JPG]]
	== Construct : Forward construction ==		== Construct : Forward construction ==

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Revision as of 14:26, 21 October 2007

The aim of the Molecular biology work was to construct the backbone for SMB genomic cassette:

In order to construct this genomic cassette, we have considered 2 solutions:

• 1)Either the construct is cloned on a plasmid. Then inserted in the genome strain of a dapA-, ftsK-(ts) E.coli strain. This was the initial strategy we adopted then subsequently abandoned (see here why LIEN).
• 2)Or the cassette is sequentially assembled, in the genome, around the essential gene selected (ftsK in our case).

We have chosen the second strategy. The different steps of this process are as follow:

2 plasmid born constructs were generated. These are termed the UP “upstream construct” & the DP “downstream construct”:

Upstream construct:

Downstream construct :

As their names indicate, these constructs are to be inserted upstream and downstream of ftsK in the genome. We chose to insert these constructs using a strategy described by Wanner et al.

We have yet to insert the constructs in the genome. This will be done sequentially:

Contents 1 Construct : Forward construction 2 Construct : Backward construct 2.1 Intermediate construct 2.2 Backward construct with DapA E.Coli 2.3 Backward construct with DapA Subtilis 3 Construct : recombinaison rate measurement

Construct : Forward construction

Construct : Backward construct

Intermediate construct

Backward construct with DapA E.Coli

Backward construct with DapA Subtilis

Construct : recombinaison rate measurement