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Team:UC Davis - 2012.igem.org

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Intro

Rational engineering targets certain chromosomal regions for manipulation so the organism will express a certain gene without a plasmid. It involves extrapolating knowledge from plasmid-based genetic experiments and applying them with electroporation or similar methods. The chromosomal expression of genes improves the efficiency of the production of the enzymes because there is no longer a cellular agenda and a human agenda for the cell [Danchin].

What we're doing

We previously learned that the strain we had received from Barcelona possessed the ability to decompose ethylene glycol to glycolate via the enzymes glycolaldehyde reductase and glycolaldehyde dehydrogenase. Our goal was to reproduce this ability with plasmids expressed in DH5α and MG1655, two ordinary E. coli strains that cannot degrade ethylene glycol. We devised two approaches to achieve this design using psb1A3. Our first procedure involves a polycistronic system, with two genes under the control of one promoter. We will have two variants of the plasmid, one with an inducible pBAD promoter and one with the constitutive J23101 promoter.

Our second approach separates the genes, allowing us to see if the genes can be expressed more efficiently when they are under the control of one promoter each. The separation also permits us to induce one promoter and therefore express one gene at a time. With the genes expressed independently, we are able to control the production of each enzyme and ensure equal amounts are expressed. The glycolaldehyde reductase enzyme will be under the control of the pBAD promoter; the glycolaldehyde dehydrogenase enzyme will be under the control of the pLAC promoter. Because we are employing the lac promoter, we must have the lacI operon to act as the repressor. The diagrams below depict the cassette orientation within each plasmid. For each of these set-ups, we will use restriction enzymes, gel purifications, and then ligations to piece together each sub-construct. The process is lengthy in time because of the time involved for transformations, liquid cultures, and enzymatic digests.

References

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Team:UC Davis/Project/Our Strain

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