Difference between revisions of "Team:Calgary/Glycolysis"

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     In order to utilize acetyl-coA, which is a product downstream of the glycolysis pathway, a naturally occurring operon in R. eutropha was used. The operon exists in the order PhaC, PhaA, and PhaB. The bacteria uses this operon to convert its excess carbon source into polyhydroxybutyrate (PHB). R. eutropha stores PHB as an energy source <source>. The  PhaC, PhaA, and PhaB gene expression leads to formation of pha synthase, acetoacetyl-CoA reductase, and 3-ketothiolase <source>. These enzymes play role in converting acetyl-CoA into acetoactyl-CoA, which is converted to (R)-3-hydroxybutyryl-CoA. Finally, this product is converted to PHB <source>.
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     In order to utilize acetyl-coA, which is a product downstream of the glycolysis pathway, a naturally occurring operon in R. eutropha was used. The operon exists in the order PhaC, PhaA, and PhaB. The bacteria uses this operon to convert its excess carbon source into polyhydroxybutyrate (PHB). R. eutropha stores PHB as an energy source (source). The  PhaC, PhaA, and PhaB gene expression leads to formation of pha synthase, acetoacetyl-CoA reductase, and 3-ketothiolase (source). These enzymes play role in converting acetyl-CoA into acetoactyl-CoA, which is converted to (R)-3-hydroxybutyryl-CoA. Finally, this product is converted to PHB (source).
 
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The naturally existing operon PhaCAB in R. eutropha is known to produce PHB. However, literature has shown that the rearrangement of the operon to PhaCBA results in higher production of PHB because PhaB results in …(add more info from paper-> talk about molecular weight as well) <source>. Hence, we decided to change the gene order from PhaCAB to PhaCBA. The operon rearrangement will lead to relatively  higher expression of PhaB. We know from literature that PhaB is important for (add more info from paper and Imperial’s modelling…) <source>. In addition to rearranging the gene order, the operon sequence was codon optimized to function in E. coli.  
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    The naturally existing operon PhaCAB in R. eutropha is known to produce PHB. However, literature has shown that the rearrangement of the operon to PhaCBA results in higher production of PHB because PhaB results in …(add more info from paper-> talk about molecular weight as well) (source). Hence, we decided to change the gene order from PhaCAB to PhaCBA. The operon rearrangement will lead to relatively  higher expression of PhaB. We know from literature that PhaB is important for (add more info from paper and Imperial’s modelling…) (source). In addition to rearranging the gene order, the operon sequence was codon optimized to function in E. coli.  
  
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<insert CAB gene construct image> <insert CBA gene construct image>
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(insert CAB gene construct image) (insert CBA gene construct image)
 
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Our gene construct was placed under an inducible promoter (lacZ, lacY). The construct was inserted into pET29(b)+ vector, which contains the promoter and kan resistant gene. The chassis used for our experiments was E. coli (BL21) because of its better ability in expression of proteins <source>. (Add more reasons for why e. Coli was used) The operon was induced using IPTG at different concentrations.
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    Our gene construct was placed under an inducible promoter (lacZ, lacY). The construct was inserted into pET29(b)+ vector, which contains the promoter and kan resistant gene. The chassis used for our experiments was E. coli (BL21) because of its better ability in expression of proteins (source). (Add more reasons for why e. Coli was used) The operon was induced using IPTG at different concentrations.
 
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Results. …
 
Results. …
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<link to parts characterization>
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(link to parts characterization)
 
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Revision as of 06:54, 13 October 2017

Header

Glycolysis

Aim

In order to utilize acetyl-coA, which is a product downstream of the glycolysis pathway, a naturally occurring operon in R. eutropha was used. The operon exists in the order PhaC, PhaA, and PhaB. The bacteria uses this operon to convert its excess carbon source into polyhydroxybutyrate (PHB). R. eutropha stores PHB as an energy source (source). The PhaC, PhaA, and PhaB gene expression leads to formation of pha synthase, acetoacetyl-CoA reductase, and 3-ketothiolase (source). These enzymes play role in converting acetyl-CoA into acetoactyl-CoA, which is converted to (R)-3-hydroxybutyryl-CoA. Finally, this product is converted to PHB (source).

Operon rearrangement

The naturally existing operon PhaCAB in R. eutropha is known to produce PHB. However, literature has shown that the rearrangement of the operon to PhaCBA results in higher production of PHB because PhaB results in …(add more info from paper-> talk about molecular weight as well) (source). Hence, we decided to change the gene order from PhaCAB to PhaCBA. The operon rearrangement will lead to relatively higher expression of PhaB. We know from literature that PhaB is important for (add more info from paper and Imperial’s modelling…) (source). In addition to rearranging the gene order, the operon sequence was codon optimized to function in E. coli. (insert CAB gene construct image) (insert CBA gene construct image)

Chassis and vector

Our gene construct was placed under an inducible promoter (lacZ, lacY). The construct was inserted into pET29(b)+ vector, which contains the promoter and kan resistant gene. The chassis used for our experiments was E. coli (BL21) because of its better ability in expression of proteins (source). (Add more reasons for why e. Coli was used) The operon was induced using IPTG at different concentrations.

Media/Culture composition & conditions

(rationale goes here)

Calgary 2017 vs. Imperial 2013

Results. … (link to parts characterization)