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 . The PhaC, PhaA, and PhaB gene expression leads to formation of pha synthase, acetoacetyl-CoA reductase, and 3-ketothiolase . 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 .

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) . 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…) . In addition to rearranging the gene order, the operon sequence was codon optimized to function in E. coli.

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 . (Add more reasons for why e. Coli was used) The operon was induced using IPTG at different concentrations.

Media/Culture composition & conditions

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Calgary 2017 vs. Imperial 2013

Results. …