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Team:UC Davis/Project/Catalyst
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Modules
LC-Cutinase and Initial PET Degradation
Design
We had the LC-Cutinase gene synthesized with a pelB leader sequence and a 6-his tag (the full construct being labeled as Bba_K936014). The pelB leader sequence is a protein tag that directs the attached protein to the periplasmic space and is removed by a pelB peptidase once in the periplasm. We used this tag as a method of protein secretion as it had been reported that once LC-cutinase was brought to the membrane, a leakage occurs that helps the catalyst to be secreted into the extracellular matrix [1]. This secretion is desirable for both the purification of the enzyme and for future applications where the bacterial culture would be directly incubated with the PET. The his-tag was included at the end of the sequence for additional purification purposes and to conduct experiments that would determine where the protein goes after production (i.e. extracellular space or inside the cell). We have two different constructs expressing this part: one with a constitutive promoter (Bba_J23101) and one with the inducible pBad promoter (Bba_K206000) allowing for induction of the cutinase gene.
Experiments
We have designed and conducted three different experiments to determine (1) if the pelB tag is working to secrete the catalyst, (2) if the LC-cutinase protein is displaying its expected esterase behavior, and (3) if the protein is capable of breaking down PET from various sources.
First, to determine whether or not pelB was transporting the cutinase enzyme outside of the cell, we conducted an experiment in which we separated the media and whole cell portions of cultures expressing the gene and ran the samples on a western blot to determine which had a higher abundance of the his-tagged cutinase. The details of this experiment can be found on the protocols page under "Cutinase Expression and Western Blot". Unfortunately, we have had few successful westerns due to excessive background on the transfer. We have spent much time trying to resolve the issue and do not have conclusive data as to where pelB is transporting the catalyst.
Second, PNPB
Ethylene Glycol Modules
Glycolaldehyde reductase (DL-1,2-propanediol oxidoreductase) is normally an anaerobic protein that reduces L-lactaldehyde into L-1,2-propanediol, an excreted product. In addition, a mutant from the paper aforementioned was able to live on L-1,2-propanediol as a sole carbon source. Not only are there mutants that live on the L-1,2-propanediol, but there are also mutants selected for growth on ethylene glycol. We are working to mutate the reductase to work aerobically, rather than anaerobically [3].
In contrast to the reductase, glycolaldehyde dehydrogenase is an aerobic protein that oxidizes glycolaldehyde further to glycolate. The glycolate will be used further downstream in metabolism to provide the carbon source for the E. coli to live.
We are using these enzymes polycistronically with the Bba_J23101 and Bba_K206000 promoters to see the difference in overproduction of the enzymes and modulating the production. Our modular efforts in plasmids will eventually be applied toward a rational strain engineering approach, where we manipulate the MG1655 chromosome to optimize the degradation of ethylene glycol.
References
2. 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.
3. Lu, Zhe, Elisa Cabiscol, Nuria Obradors, Jordi Tamarit, Joaquim Ros, Juan Aguilar, and E.C.C. Lin. "Evolution of an Escherichia coli Protein with Increased Resistance to Oxidative Stress." Journal of Biological Chemistry. 273.14 (1998): n. page. Print.
