Difference between revisions of "Team:Calgary/Synthesis"

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<h3>Chassis and vector</h3>
 
<h3>Chassis and vector</h3>
  
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<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 kanamycin-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 (Black & DiRusso, 1994). 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.
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<p>Our genetic constructs were placed under a T7 IPTG-inducible promoter (lacZ, lacY in the pET29(B)+ vector, which contains the gene for kanamycin resistance. The chassis used for our experiments was <i>E. coli</i> BL21(DE3), whose genome contains the sequence for T7 RNA polymerase to allow for transcription of our constructs. 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 (Black & DiRusso, 1994).
 
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<h3> Genes and Choice of pathways</h3>
 
<h3> Genes and Choice of pathways</h3>
  
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<p>Hiroe et al. (2012) 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.</p>
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<p>Hiroe <i>et al.</i> (2012) investigated the effect that gene order of the <i>phaCAB</i> operon had on the PHB molecular yield. They found that the highest PHB yield was found when the operon's genes were ordered as <i>phaCBA</i>. (More details can be found <a href="https://2017.igem.org/Team:Calgary/Glycolysis">here</a>.) Therefore, we decided to use this gene order to maximize PHB production.</p>
  
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<p>In addition, Davis et al. (2008) showed that expression of phaC1 and phaJ from PHA-producing bacteria such as <i>Pseudomonas aeruginosa</i> produced the copolymer PHB and medium chain length PHA. The phaC1 gene from <i>P. aeruginosa</i> encodes PHA synthase, which regulates the final step in both the beta-oxidation and PHA synthesis pathways. (We will refer to the PHA synthesis pathway as the glycolysis pathway). This function of PHA synthase makes it one of two important enzymes in our pathway as it accepts 3-hydroxybutyryl-CoA molecules, cleaves off the CoA segment, and polymerizes the 3-hydroxybutyrate into PHB. The other key enzyme in our pathway is encoded for by phaJ4, which we selected from <i>P. aeruginosa</i> based on findings from Tsuge et al. (2011). Our research showed that the enzyme encoded for by phaJ4, enoyl-CoA hydratase, channels products of the beta-oxidation pathway into the glycolysis pathway.</p>
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<p>In addition, Davis <i>et al.</i> (2008) showed that expression of <i>phaC1</i> and <i>phaJ</i> from PHA-producing bacteria such as <i>Pseudomonas aeruginosa</i> and <i>putida</i> produced PHB and medium chain length PHAs. The <i>phaC1</i> gene from <i>P. aeruginosa</i> encodes PHA synthase, which regulates the final step in both the beta-oxidation and PHA synthesis pathways. This function of PHA synthase makes it one of two important enzymes in our pathway as it accepts 3-hydroxybutyryl-CoA molecules, cleaves off the CoA segment, and polymerizes the 3-hydroxybutyrate into PHB. The other key enzyme in our pathway is encoded for by <i>phaJ4</i>, which we selected from <i>P. putida</i> based on findings from Tsuge <i>et al.</i> (2011). Our research showed that the enzyme encoded for by <i>phaJ4</i>, enoyl-CoA hydratase, channels products of the beta-oxidation pathway into the glycolysis pathway.</p>
  
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Revision as of 05:29, 31 October 2017

Header

Synthesis

Synthesis Pathway

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:

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

Analysis of human waste

Chassis and vector

Our genetic constructs were placed under a T7 IPTG-inducible promoter (lacZ, lacY in the pET29(B)+ vector, which contains the gene for kanamycin resistance. The chassis used for our experiments was E. coli BL21(DE3), whose genome contains the sequence for T7 RNA polymerase to allow for transcription of our constructs. 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 (Black & DiRusso, 1994).

Genes and Choice of pathways

Hiroe et al. (2012) investigated the effect that gene order of the phaCAB operon had on the PHB molecular yield. They found that the highest PHB yield was found when the operon's genes were ordered as phaCBA. (More details can be found here.) Therefore, we decided to use this gene order to maximize PHB production.

In addition, Davis et al. (2008) showed that expression of phaC1 and phaJ from PHA-producing bacteria such as Pseudomonas aeruginosa and putida produced PHB and medium chain length PHAs. The phaC1 gene from P. aeruginosa encodes PHA synthase, which regulates the final step in both the beta-oxidation and PHA synthesis pathways. This function of PHA synthase makes it one of two important enzymes in our pathway as it accepts 3-hydroxybutyryl-CoA molecules, cleaves off the CoA segment, and polymerizes the 3-hydroxybutyrate into PHB. The other key enzyme in our pathway is encoded for by phaJ4, which we selected from P. putida based on findings from Tsuge et al. (2011). Our research showed that the enzyme encoded for by phaJ4, enoyl-CoA hydratase, channels products of the beta-oxidation pathway into the glycolysis 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.

Davis, R., Anilkumar, P.K., Chandrashekar, A. & Shamala, T.R. (2008). Biosynthesis of polyhydroxyalkanoates co-polymer in E. coli using genes from Pseudomonas and Bacillus. Antonie Van Leeuwenhoek. 94: 207-16.

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.