Team:Nevada/Project
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To integrate the GLF gene into the Synechocystis genome, we have decided to use an alternative insertion site. In this case we will insert the GLF overexpression gene cassette along with a chloramphenicol resistance cassette (CamR) into the coding region of the thiamin monophosphate pyrophosphorylase (ThiE) gene. The rationale for creating a ThiE knockout mutant is that it will lead to the creation of an auxotophic mutant that will only survive when the grown in the presence of thiamin (Vitamin B1). Therefore, the transgenic Synechocystis will not be able to survive outside laboratory and the chances of environmental contamination will be decreased. We will simultaneously create the ThiE mutant and the GLF overexpressing line by creating an ThiE gene replacement construct in which the GLF overexpression gene cassette and a chloramphenicol resistance gene cassette are inserted into the coding region of a subcloned ThiE gene. This dysfunctional ThiE construct will then be transformed into Synechocystis and through homologous recombination, replace the native ThiE gene. | To integrate the GLF gene into the Synechocystis genome, we have decided to use an alternative insertion site. In this case we will insert the GLF overexpression gene cassette along with a chloramphenicol resistance cassette (CamR) into the coding region of the thiamin monophosphate pyrophosphorylase (ThiE) gene. The rationale for creating a ThiE knockout mutant is that it will lead to the creation of an auxotophic mutant that will only survive when the grown in the presence of thiamin (Vitamin B1). Therefore, the transgenic Synechocystis will not be able to survive outside laboratory and the chances of environmental contamination will be decreased. We will simultaneously create the ThiE mutant and the GLF overexpressing line by creating an ThiE gene replacement construct in which the GLF overexpression gene cassette and a chloramphenicol resistance gene cassette are inserted into the coding region of a subcloned ThiE gene. This dysfunctional ThiE construct will then be transformed into Synechocystis and through homologous recombination, replace the native ThiE gene. | ||
+ | |||
+ | ''' | ||
+ | == '''Construct Design''' == | ||
+ | '''<br> | ||
+ | |||
+ | https://static.igem.org/mediawiki/2011/f/f0/AGP_INV_UNR1.jpg<br> | ||
+ | |||
+ | ''' | ||
== '''Gene Construct Components''' == | == '''Gene Construct Components''' == |
Revision as of 17:11, 26 September 2011
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