Team:Nevada/Project
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- | + | ''' | |
- | ''Synechocystis | + | == Task 2) Engineer ''Synechocystis'' to secrete hexose sugar to the medium == |
+ | ''' | ||
+ | <br> | ||
+ | Synechocystis lacks the ability to secrete hexose sugars. Therefore it was necessary to introduce a sugar transporter through genetic engineering. Previous studies (Nedierholtmeyer et al (2010) Applied and Environmental Microbiology (2010) 76: 3462) showed that by expressing the Zymonomas mobilis glucose facilitative transporter (GLF) gene in Synechococcus, glucose and fructose were detected in the culture medium at levels as high as 30 M and 150 M respectively. Based on these results we decided to express GLF in Synechocystis. Our hope is that by expressing GLF in the AGP KO/INV overexpressing line, we will be able to produce enough glucose to the surrounding medium to support the E. coli biofuel production. | ||
+ | |||
+ | ''' | ||
+ | == Approach == | ||
+ | '''<br> | ||
+ | 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. | ||
+ | |||
+ | |||
+ | == '''Gene Construct Components''' == | ||
+ | <br> | ||
+ | '''ThiE Gene:''' The sll0635 open reading frame was PCR amplified from total Synechocystis PCC6803 genomic DNA and subcloned into pSB1a3. Forward and reverse primers were designed with 5’ extensions containing iGEM prefix and suffix sequences.<br> | ||
+ | |||
+ | '''Glucose facilitative transporter (GLF) Gene+double terminator:''' The GLF gene was synthesized based on the sequence of a Zymonomas mobilis invertase gene described by Neiderholtmeyer et al. (Applied and Environmental Microbiology (2010) 76: 3462) which was codon optimized for expression in cyanobacteria. The gene was also synthesized with a double transcriptional terminator designed from sequences described for iGEM part BBa_B0005.<br> | ||
+ | |||
+ | '''petBD promoter+RBS:''' The strong light inducible petBD promoter will be used to drive the expression of invertase gene in Synechocystis. The petBD+RBS (BBa_K390015) was generously provided to us by the Utah State iGEM team.<br> | ||
+ | |||
+ | '''Chloramphenicol resistance cassette (CamR):''' The chloramphenicol resistance cassette was PCR amplified from pSB1C3. This cassette includes the chloramphenicol acetyltransferase gene with its own constitutive promoter and transcriptional terminator. This cassette is frequently used to select for positive transformants in Synechocystis | ||
== | == |
Revision as of 17:04, 26 September 2011
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