Revision as of 04:46, 31 October 2017

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Synthesis

Overview

The overarching goal for the synthesis component of the project was to produce poly-3-hydroxybutyrate (PHB) by utilizing the nutrients present in human waste. In order to accomplish this, we:

analyzed human waste and chose organic compounds to use as feedstocks for our bacteria, and
researched and optimized efficient pathways to turn relevant components of human waste (acetyl-CoA and volatile fatty acids) into PHB

Analysis of human waste

Choice of pathways

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). The bacterium, E. coli, is known to adapt well to both aerobic and anaerobic conditions and grow quickly given an adequate carbon source. Being a popular model organism, its metabolic pathways are well-studied and, thus, provided us with an extensive array of identified molecules and structural components to manipulate. We also found that E. colihas a better ability to express proteins (source). The operon was induced using IPTG at different concentrations.

Hiroe et al. investigated the effect that gene order of the phaCAB operon had on the PHA molecular weight and yield. They found that the molecular weight was most balanced in the strain harboring phaBCA expression plasmid. However, the highest PHA yield was found in the order phaCBA. Therefore, we decided to use this gene order to maximize PHA production.

In addition, Davis et al. showed that expression of phaC1 and phaJ from PHA-producing bacteria such as Pseudomonas aeruginosa produced the copolymer PHB and medium chain length PHA. We selected phaJ4 from P. aeruginosa based on findings from Tsuge et al. Our research showed that the protein encoded for by phaJ4 (enoyl-CoA hydratase) channeled products of the Beta-Oxidation Pathway into the PHA Synthesis Pathway.

WORKS CITED

Black, P.N. & DiRusso, C.C. (1994). Molecular and biochemical analyses of fatty acid transport, metabolism, and gene regulation in Escherichia coli. Biochimica et Biophysica Acta. 1210: 123-145.

Hiroe, A., Tsuge, K., Nomura, C.T., Itaya, M. & Tsuge, T. (2012). Rearrangement of gene order in the phaCAB operon leads to effective production of ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] in genetically engineered Escherichia coli. Applied and Environmental Microbiology. 78: 3177–3184.

Sato, S., Kanazawa H. & Tsuge, T. (2011). Expression and characterization of (R)-specific enoyl coenzyme A hydratases making a channeling route to polyhydroxyalkanoate biosynthesis in Pseudomonas putida. Applied Microbiology Biotechnology. 90: 951-9.

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 <h2>Chassis and vector</h2>
-<p>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 <i>E. coli</i> (BL21). The bacterium, <i>E. coli</i>, is known to adapt well to both aerobic and anaerobic conditions and grow quickly given an adequate carbon source. Being a popular model organism, its metabolic pathways are well-studied and, thus, provide us with an extensive array of identified molecules and structural components to manipulate. <i>E. coli</i> also has a better ability to express proteins (source). The operon was induced using IPTG at different concentrations.
+<p>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 <i>E. coli</i> (BL21). The bacterium, <i>E. coli</i>, is known to adapt well to both aerobic and anaerobic conditions and grow quickly given an adequate carbon source. Being a popular model organism, its metabolic pathways are well-studied and, thus, provided us with an extensive array of identified molecules and structural components to manipulate. We also found that <i>E. coli</i>has a better ability to express proteins (source). The operon was induced using IPTG at different concentrations.
 </p>