IGEM:IMPERIAL/2006/project/popsblocker/Implementation

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(β†’Primers)
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[[Image:Cre.PNG]]
[[Image:Cre.PNG]]
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==Ligation strategy==
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*Apart from ligating into the necessary plasmid at the end, there are no ligation steps involved in making this device. PCR and PCR fusion are being are used because of the unusual design of the device, in so far as both strands have coding regions. The link below explains the method by which we will use PCR to create the device.
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[[Image:Cre3.PNG]]
[[Image:Cre3.PNG]]
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Stop Codons
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*[http://openwetware.org/images/5/5a/Cre_Pops_Blocker.ppt Proposed Implementation of Cre-LoxP system (ppt)]
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I Have included a stop codon between lox66 and B0021 to prevent the e-coli cells from translating past the first lox site. This reduces the load that this part will place on the E.coli cell so increases it’s stability in vivo. I checked the E-coli codon usage database on NCBI and found that TGA was by far the most commonly used in nature.
 
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Frameshift
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==Considerations==
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The Lox sites are 34bp long therefore if this part is placed after an RBS it will induce a frameshift in the protein produced, therefore creating a mangled protein. I added two random bases to the start of the lox site to prevent a frameshift.
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'''Stop Codons'''
 +
A stop codon has been included between Lox66 and B0021 to prevent the ''E.Coli'' cells from translating past the first Lox site. This reduces the load that this part will place on the E.coli cell so increases its stability ''in vivo''. TGA is by far the most commonly used stop codon in ''E.Coli''.
 +
 +
'''Frameshift'''
 +
 +
The Lox sites are 34bp long therefore if this part is placed after an RBS it will induce a frameshift in the protein produced, therefore creating a mangled protein. Two random bases were added to the start of the Lox site to prevent a frameshift.
-
==Ligation strategy==
 
-
*Apart from ligating into the necessary plasmid at the end, there are no ligation steps involved in making this device. PCR and PCR fusion are being are used because of the unusual design of the device, in so far as both strands have coding regions. The link below explains the method by which we will use PCR to create the device.
 
-
 
-
*[http://openwetware.org/images/5/5a/Cre_Pops_Blocker.ppt Proposed Implementation of Cre-LoxP system (ppt)]
 
==Primers==
==Primers==
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[http://openwetware.org/images/5/5b/Cre_PCR.doc Design of primers and method of construction]
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[http://openwetware.org/images/5/58/Redoing_Cre2a.doc Design of primers and method of construction]
[http://openwetware.org/images/b/ba/A112-705-Cre_Version_2_1.pdf Commercially available Cre plasmid]
[http://openwetware.org/images/b/ba/A112-705-Cre_Version_2_1.pdf Commercially available Cre plasmid]
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[http://openwetware.org/images/5/58/Redoing_Cre2a.doc Design of primers and method of construction]
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==WetLab work entries==
==WetLab work entries==

Revision as of 16:50, 29 October 2006


Image:Cre.PNG

Contents

Ligation strategy

  • Apart from ligating into the necessary plasmid at the end, there are no ligation steps involved in making this device. PCR and PCR fusion are being are used because of the unusual design of the device, in so far as both strands have coding regions. The link below explains the method by which we will use PCR to create the device.

Image:Cre3.PNG


Considerations

Stop Codons

A stop codon has been included between Lox66 and B0021 to prevent the E.Coli cells from translating past the first Lox site. This reduces the load that this part will place on the E.coli cell so increases its stability in vivo. TGA is by far the most commonly used stop codon in E.Coli.

Frameshift

The Lox sites are 34bp long therefore if this part is placed after an RBS it will induce a frameshift in the protein produced, therefore creating a mangled protein. Two random bases were added to the start of the Lox site to prevent a frameshift.


Primers

Design of primers and method of construction

Commercially available Cre plasmid


WetLab work entries

Jonny Wells
Kirsten Jensen

(08/06 - 10/06)

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