Paris/ConstructionProcess

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{{Template:Paris_menu}}
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== Overview ==
== Overview ==
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The aim of the Molecular biology work was to construct the backbone for SMB genomic cassette:
 
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[[Image:Paris_FinalConstruct.JPG|400px]]
 
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In order to construct this genomic cassette, we have considered 2 solutions:
+
Through classical molecular biology techniques, we aimed to construct the following genomically inserted cassette
-
* 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).
+
[[Image:Paris_FinalConstruct2.JPG|550px]]
-
* 2. Or the cassette is sequentially assembled, in the genome, around the essential gene selected (ftsK in our case).  
+
 
 +
This full construct has yet to be assembled in the genome.
 +
 
 +
As can be seen on this schematic representation of the SMB genomic backbone cassette, at basal genomic state (in Germ line cells):
 +
 
 +
*pTet promoter drives the expression of gfp.
 +
*The expression of ftsK is controlled by its natural promoter.
 +
*ftsK is isolated from pTet promoter by the intercalation of Terminator (B0015 terminator).
 +
*dapA gene is in a dormant state since it lacks a promoter to drive its transcription.
 +
 
 +
 
 +
Upon G to S differentiation, the following genomic reassembly should take place:
 +
constructed. These are termed the “'''upstream construct'''” & the “'''downstream construct'''”:
 +
 
 +
[[Image:Paris_Recombination scheme.JPG|600px]]
 +
 
 +
 
 +
 
 +
Cre mediated lox recombination should lead to:
 +
 
 +
*excision of ftsK gene from the genome on a circular unreplicative DNA molecule. We ignore how much time such a DNA is stable in a cell.
 +
*placing dapA gene under the control of pTet promoter. This should lead to dapA expression, & hopefully to DAP synthesis.
 +
 
 +
 
 +
 
 +
 
 +
----
 +
 
 +
 
 +
 
 +
In order to construct this genomic cassette, we have considered two technical solutions:
 +
 
 +
 
 +
1) Either the full construct is cloned on a plasmid and inserted in the genome of a ''dapA-'', ''ftsK-''(ts)  E.coli strain. This was the initial strategy we adopted then subsequently abandoned. cloning ftsZ gene, which we had initially selected as the essential gene in our construct, has proven to be quite problematic.  
 +
2) Or the cassette is sequentially assembled, in the genome, around the essential gene selected (ftsK in our case).  
 +
 
 +
The second strategy does not require cloning ftsK gene & does not require using an ''ftsK'' mutant.
 +
 
 +
We have chosen the second strategy mentioned above to generate the SMB genomic backbone construct. The different steps of this process are as follow:
 +
 
 +
2 plasmid born constructs were generated. These are termed the “upstream construct” & the “downstream construct”:
 +
 
 +
 
 +
Upstream construct:
-
We have chosen the second strategy. The different steps of this process are as follow:
 
-
2 plasmid born constructs were constructed. These are termed the UP “'''upstream construct'''” & the DP “'''downstream construct'''”:
 
'''Upstream construct:'''
'''Upstream construct:'''
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[[Image:Paris_UpstreamConstruct.JPG|400px]]
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[[Image:Paris_UpstreamConstruct.JPG|500px]]
 +
 
 +
[[Upstream construct|SEE HERE]] how the upstream construct sequentially assembled on a plasmid.
'''Downstream construct:'''
'''Downstream construct:'''
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[[Image:Paris_DownstreamConstruct.JPG|400px]]
+
[[Image:Paris_DownstreamConstruct.JPG|500px]]
-
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.
+
[[Downstream construct|SEE HERE]] how the downstream construct was sequentially assembled
-
We have yet to insert the constructs in the genome. This will be done sequentially:
 
-
[[Image:Paris_UpstreamInsertion.JPG|400px]]
+
As their names indicate, these constructs are to be inserted upstream and downstream of ftsK gene in the genome of a ''dapA''- strain.
 +
We chose to insert these constructs using a strategy described by Datsenko KA. & Wanner BL. (PNAS 2000)
-
[[Image:Paris_FlpRec.JPG|400px]]
 
 +
We have yet to insert the constructs in the genome. This will be done sequentially:
 +
In a first step the downstream construct is amplified using oligonucteotides baring sequences homologous to those downstream of  ftsK. The PCR product is used to transform a dapA- E.coli strain.
-
[[Image:Paris_DownstreamInsertion.JPG|600px]]
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[[Image:Paris_Downstream Insertion.JPG|500px]]  
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== Constructs ==
 
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=== Upstream construct ===
 
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<html>
 
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<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=6,0,29,0" width="800" height="600">
 
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<param name="movie" value="http://poupitoupou.free.fr/Construct_FW.swf">
 
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<param name="quality" value="high">
 
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<embed src="http://poupitoupou.free.fr/Construct_FW.swf" quality="high" pluginspage="http://www.macromedia.com/go/getflashplayer" type="application/x-shockwave-flash" width="500" height="750"></embed></object>
 
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</html>
 
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=== Downstream construct ===
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The resistance cassette frt-CmR-frt carried by the downstream construct allows screening for the event of genomic insertion of the construct. CmR gene confers resistance to the antibiotic chloramphenicol.
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==== Intermediate construct ====
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<br>
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<html>
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<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=6,0,29,0" width="800" height="600">
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<param name="movie" value="http://poupitoupou.free.fr/Construct_Intermediate.swf">
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<param name="quality" value="high">
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<embed src="http://poupitoupou.free.fr/Construct_Intermediate.swf" quality="high" pluginspage="http://www.macromedia.com/go/getflashplayer" type="application/x-shockwave-flash" width="400" height="500"></embed></object>
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</html>
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====  Backward construct with DapA E.Coli ====
 
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<br>
 
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<html>
 
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<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=6,0,29,0" width="800" height="600">
 
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<param name="movie" value="http://poupitoupou.free.fr/Construct_BW_DapE.swf">
 
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<param name="quality" value="high">
 
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<embed src="http://poupitoupou.free.fr/Construct_BW_DapE.swf" quality="high" pluginspage="http://www.macromedia.com/go/getflashplayer" type="application/x-shockwave-flash" width="450" height="350"></embed></object>
 
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</html>
 
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====  Backward construct with DapA Subtilis ====
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Subsequently, the Chloramphenicol resistance gene is eliminated by FRT recombination. This reaction is to be catalysed by the site specific recombinase: Flp.
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<br>
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<html>
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<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=6,0,29,0" width="800" height="600">
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<param name="movie" value="http://poupitoupou.free.fr/Construct_BW_DapS.swf">
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-
<param name="quality" value="high">
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<embed src="http://poupitoupou.free.fr/Construct_BW_DapS.swf" quality="high" pluginspage="http://www.macromedia.com/go/getflashplayer" type="application/x-shockwave-flash" width="600" height="550"></embed></object>
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</html>
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=== Recombinaison rate measurement ===
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-
<br>
+
 
-
<html>
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[[Image:Paris_Flp.JPG|500px]]
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<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=6,0,29,0" width="800" height="600">
+
 
-
<param name="movie" value="http://poupitoupou.free.fr/Construct_MRR.swf">
+
 
-
<param name="quality" value="high">
+
The upstream construct can then be inserted based on the same strategy.
-
<embed src="http://poupitoupou.free.fr/Construct_MRR.swf" quality="high" pluginspage="http://www.macromedia.com/go/getflashplayer" type="application/x-shockwave-flash" width="500" height="600"></embed></object>
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-
</html>
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 +
[[Image:Paris_UpstreamInsertion.JPG|700px]]

Latest revision as of 03:08, 27 October 2007


Overview

Through classical molecular biology techniques, we aimed to construct the following genomically inserted cassette

Paris FinalConstruct2.JPG

This full construct has yet to be assembled in the genome.

As can be seen on this schematic representation of the SMB genomic backbone cassette, at basal genomic state (in Germ line cells):

  • pTet promoter drives the expression of gfp.
  • The expression of ftsK is controlled by its natural promoter.
  • ftsK is isolated from pTet promoter by the intercalation of Terminator (B0015 terminator).
  • dapA gene is in a dormant state since it lacks a promoter to drive its transcription.


Upon G to S differentiation, the following genomic reassembly should take place: constructed. These are termed the “upstream construct” & the “downstream construct”:

Paris Recombination scheme.JPG


Cre mediated lox recombination should lead to:

  • excision of ftsK gene from the genome on a circular unreplicative DNA molecule. We ignore how much time such a DNA is stable in a cell.
  • placing dapA gene under the control of pTet promoter. This should lead to dapA expression, & hopefully to DAP synthesis.





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


1) Either the full construct is cloned on a plasmid and inserted in the genome of a dapA-, ftsK-(ts) E.coli strain. This was the initial strategy we adopted then subsequently abandoned. cloning ftsZ gene, which we had initially selected as the essential gene in our construct, has proven to be quite problematic. 2) Or the cassette is sequentially assembled, in the genome, around the essential gene selected (ftsK in our case).

The second strategy does not require cloning ftsK gene & does not require using an ftsK mutant.

We have chosen the second strategy mentioned above to generate the SMB genomic backbone construct. The different steps of this process are as follow:

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


Upstream construct:


Upstream construct: Paris UpstreamConstruct.JPG

SEE HERE how the upstream construct sequentially assembled on a plasmid.

Downstream construct: Paris DownstreamConstruct.JPG

SEE HERE how the downstream construct was sequentially assembled


As their names indicate, these constructs are to be inserted upstream and downstream of ftsK gene in the genome of a dapA- strain.

We chose to insert these constructs using a strategy described by Datsenko KA. & Wanner BL. (PNAS 2000)


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

In a first step the downstream construct is amplified using oligonucteotides baring sequences homologous to those downstream of ftsK. The PCR product is used to transform a dapA- E.coli strain.

Paris Downstream Insertion.JPG


The resistance cassette frt-CmR-frt carried by the downstream construct allows screening for the event of genomic insertion of the construct. CmR gene confers resistance to the antibiotic chloramphenicol.


Subsequently, the Chloramphenicol resistance gene is eliminated by FRT recombination. This reaction is to be catalysed by the site specific recombinase: Flp.


Paris Flp.JPG


The upstream construct can then be inserted based on the same strategy.


Paris UpstreamInsertion.JPG