Project/PlcR Promoter/

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== PlcR Promoter ==
== PlcR Promoter ==
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Revision as of 09:47, 19 October 2010


Contents

PlcR Promoter


Background


Sequence Development and Annotation

In the same way that we assembled the Lac/Ara-1 promoter from primer-primer adhesion and their subsequent extension, the PlcR promoter was assembled in the same fashion. See the Lac1/AraC Promoter for the full assembly details.

Promoter Assembly

The forward primer used in assembly is:
Plcr forward promoter.JPG
The sequence in red is the EcoR1 restriction site with that in blue being the Xba1 restriction site. The region in purple are the bases that the forward and reverse primers overlap with one another.

The reverse primer used in assembly is:
Plcr reverse promoter.JPG
The sequence in green is a Pst1 restriction site and that in orange is the Spe1 restriction site. The region in purple is the sequence of shared overlap with the forward primer. As can be seen, there have been randomised nucleotides introduced so as to create a library of varying promoters. This was performed so as to increase the stochastic nature associated with the promoter efficacy. According to the IUPAC nomenclature, Y indicates either a C or a T while W indicates either an A or a T.

The entire PlcR promoter sequence is:
Plcr promoter full.JPG
As can be seen, the 'g' in red resulted from the insertion of a C and the 't' in green from the insertion of an A.

The Package

The plasmid map, annotated with appropriate restriction sites and sizes depicts the PlcR promoter ligated into PSB1C3.
PlcR in PSB1C3.JPG

Testing and Validation

The following confirmation of the successful ligation of the PlcR promoter into PSB1C3 is in a 2% (m/v) agarose gel.
Plcr confirmation gel.JPG
The above gel indicates the PlcR promoter (135bp) and the linearised PSB1C3 plasmid (2072). The fragments were isolated by digestion with EcoR1 and Pst1.


References