Team:Nevada/Project
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Strongtruong (Talk | contribs) (→2) Engineer Synechocystis to secrete hexose sugar to the medium) |
Strongtruong (Talk | contribs) (→2) Engineer Synechocystis to secrete hexose sugar to the medium) |
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''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. | ''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> | + | == |
- | + | '''Approach''' == | |
- | 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. | + | <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> | |
- | + | ||
- | '''Gene Construct Components'''<br> | + | |
'''ThiE Gene:''' The slr1176 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> | '''ThiE Gene:''' The slr1176 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> | ||
Revision as of 05:47, 26 September 2011
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