Difference between revisions of "Team:Calgary/Glycolysis"

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<br><h2>Aim</h2>
 
<br><h2>Aim</h2>
  
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<p><p>Glycolysis is native to all living cells, and uses glucose to produce pyruvate as the first step to producing energy in the form of ATP. Pyruvate is later converted to acetyl-CoA in the first step of the citric acid cycle. In <i>Ralstonia eutropha</i>, excess acetyl-coA is converted into poly(3-hydroxybuturate) (PHB) and stored as a carbon energy source (Hiroe <i>et al.</i>, 2012). <i>R. eutropha</i> accomplishes this feat with the use of three genes:
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    In order to utilize acetyl-coA, which is a product downstream of the glycolysis pathway, a naturally occurring operon in <i>R. eutropha</i> was used. The operon exists in the order phaC, phaA, and phaB. The bacteria uses this operon to convert its excess carbon source into poly[(R)-3-hydroxybutyrate] (PHB) . <i>R. eutropha</i> stores PHB as an energy source (Hiroe <i>et al.</i>, 2012). Transcription of the phaCAB operon leads to expression of the following enzymes in the order: pha synthase, acetoacetyl-CoA reductase, and 3-ketothiolase. The expression of phaA leads to expression of 3-ketothiolase that converts acetyl-coA to acetoacetyl-CoA. The acetoacetyl-CoA reductase enzyme resulting from the expression of phaB leads to conversion of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA. Finally, pha synthase leads to synthesis of PHB from (R)-3-hydroxybutyryl-CoA (Hiroe <i>et al.</i>, 2012).
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<li><i>phaA</i>, which codes for 3-ketothiolase and converts acetyl-CoA to acetoacetyl-CoA;</li>
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<li><i>phaB</i>, which codes for acetoacetyl-CoA reductase, which converts acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA, and;</li>
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<li><i>phaC</i>, which codes for pha synthase and converts (R)-3-hydroxybutyryl-CoA, to PHB.</li>
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Our goal was to express these genes in <i>E. coli</i> to produce PHB using glucose as an initial substrate.</p>
  
 
<h2>Operon rearrangement</h2>
 
<h2>Operon rearrangement</h2>

Revision as of 04:23, 31 October 2017

Header

Glycolysis

Glycolysis Pathway


Aim

Glycolysis is native to all living cells, and uses glucose to produce pyruvate as the first step to producing energy in the form of ATP. Pyruvate is later converted to acetyl-CoA in the first step of the citric acid cycle. In Ralstonia eutropha, excess acetyl-coA is converted into poly(3-hydroxybuturate) (PHB) and stored as a carbon energy source (Hiroe et al., 2012). R. eutropha accomplishes this feat with the use of three genes:

  • phaA, which codes for 3-ketothiolase and converts acetyl-CoA to acetoacetyl-CoA;
  • phaB, which codes for acetoacetyl-CoA reductase, which converts acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA, and;
  • phaC, which codes for pha synthase and converts (R)-3-hydroxybutyryl-CoA, to PHB.
Our goal was to express these genes in E. coli to produce PHB using glucose as an initial substrate.

Operon rearrangement

The naturally existing operon phaC, phaA, and phaB in R. eutropha is known to produce PHB. However, literature has shown that the rearrangement of the operon to phaC, phaB, and phaA results in higher production of PHB (Hiroe et al., 2012). Hence, we decided to change the gene order from phaCAB to phaCBA. The operon rearrangement to phaCBA will lead to relatively higher expression of phaB compared to expression in phaCAB. Hiroe et al. showed that the content of PHB is dependent on expression of phaB. However, the molecular weight of PHB was not affected by the different levels of expression of phaB. The following image shows the rearrangement of the phaCAB operon to phaCBA.

Glycolysis CAB Construct

Glycolysis CBA Construct

Figure 1. Naturally existing phaCAB operon in R. eutropha on top and rearranged operon phaCBA on bottom.

Media/Culture composition

The phaCBA operon can utilize acetic acid present in fermented synthetic feces supernatant (which is referred to as "syn poo" supernatant). In order to test our gene construct, the operon was inserted into pET29(b)+. Competent E. coli (BL21) was then transformed with the plasmid. The overnights of successfully transformed cells was then used for our experiments. The different media composition used were glucose only and fermented syn poo supernatant. The glucose only condition will show the ability of our construct to use glucose as a feedstock (positive control) and the syn poo supernatant will be used to test whether our construct can synthesize PHB from synthetic feces.

Results

The results of the experiments for our phaCBA construct is given on results page and our parts registry.

References

Hiroe A, Tsuge K, Nomura CT, 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. Appl. Environ. Microbiol. 78:3177–3184. 10.1128/AEM.07715-11.