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Team:UC Davis/Project/Catalyst
From 2012.igem.org
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<b>Design</b><br> | <b>Design</b><br> | ||
| - | We had the LC-Cutinase gene synthesized with a pelB leader sequence and a 6-his tag and labeled it <a href="http://partsregistry.org/Part:BBa_K936014">Bba_K936014</a>. The pelB leader sequence is a protein tag that directs the protein to the periplasmic space. The tag is removed by a pelB peptidase once in the periplasm. The LC-cutinase paper used a pelB tag and reported that once the cutinase was brought to the membrane, there is a leakage 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. We included a his-tag to the end of the sequence so that we could purify the cutinase for incubation with PET as well as identify where it goes after production (i.e. extracellular space or inside the cell). We have two different constructs expressing this part: one with a constitutive promoter (J23101) and one with the inducible pBad promoter (k206000)allowing for induction of the cutinase gene. | + | We had the LC-Cutinase gene synthesized with a pelB leader sequence and a 6-his tag and labeled it <a href="http://partsregistry.org/Part:BBa_K936014">Bba_K936014</a>. The pelB leader sequence is a protein tag that directs the protein to the periplasmic space (1). The tag is removed by a pelB peptidase once in the periplasm. The LC-cutinase paper used a pelB tag and reported that once the cutinase was brought to the membrane, there is a leakage 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. We included a his-tag to the end of the sequence so that we could purify the cutinase for incubation with PET as well as identify where it goes after production (i.e. extracellular space or inside the cell). We have two different constructs expressing this part: one with a constitutive promoter (J23101) and one with the inducible pBad promoter (k206000)allowing for induction of the cutinase gene. |
<center><br><img src="https://static.igem.org/mediawiki/2012/2/28/UCDavisNew_pBAD_B34_Cutinase.png"></center> | <center><br><img src="https://static.igem.org/mediawiki/2012/2/28/UCDavisNew_pBAD_B34_Cutinase.png"></center> | ||
Revision as of 05:45, 2 October 2012
UCDavis iGEM Tweets
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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 and labeled it Bba_K936014. The pelB leader sequence is a protein tag that directs the protein to the periplasmic space (1). The tag is removed by a pelB peptidase once in the periplasm. The LC-cutinase paper used a pelB tag and reported that once the cutinase was brought to the membrane, there is a leakage 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. We included a his-tag to the end of the sequence so that we could purify the cutinase for incubation with PET as well as identify where it goes after production (i.e. extracellular space or inside the cell). We have two different constructs expressing this part: one with a constitutive promoter (J23101) and one with the inducible pBad promoter (k206000)allowing for induction of the cutinase gene.
Experiments
We have designed and conducted an experiment to determine how much of the protein is being secreted and how much is remaining inside of the cell, the results of which can be found here (link coming soon).
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.
