Strain

Background

We found an E. coli mutant that is able to grow on ethylene glycol, one of the two products created during PET degradation. One goal of our project is to maximize breakdown of ethylene glycol as much as possible because it is a toxin, and also because once broken down, it can later be used in metabolism, which makes our whole project more self sustaining. The strain we found is from Barcelona, Spain, and can survive on ethylene glycol as the only carbon source [1]

What we're doing

PCR SOEing (Polymerase Chain Reaction Splicing by Overlapping Extension: As an important step in our project we needed to use a technique called PCR SOEing which is used to attach DNA fragments together without the many steps of digesting and ligating. Our two enzymes that break down ethylene glycol, glycoaldehyde reductase and glycoaldehyde dehydrogenase, needed to have the Eco, Xba, Spe or Pst restriction sites removed in order to biobrick the enzyme. So we had to look at the sequence of both enzymes and determine if they had any of the restriction site sequences. What we found was that glycoaldehyde reductase had one Pst restriction site and glcoaldehyde dehydrogenase had two Pst sites. How SOEing works: in order to do SOEing, forward and reverse primers must be designed for where you want to attach and the whole enzyme as well. This is done by PCR reactions, and the ways we assay if the DNA fragments annealed is by doing a digest with the coordinating restriction enzyme.
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

1. Boronat, Albert, Caballero, Estrella, and Juan Aguilar. “Experimental Evolution of a Metabolic Pathway for Ethylene Glycol Utilization by Escherichia coli.” Journal of Bacteriology, Vol. 153 No. 1, pp. 134-139, January 1983.

Team:UC Davis/Project/Strain

From 2012.igem.org

Strain