Difference between revisions of "Team:Calgary/Glycolysis"

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     In order to utilize acetyl-coA, which is a product downstream of the glycolysis pathway, a naturally occurring operon in R. eutropha was used. The operon exists in the order PhaC, PhaA, and PhaB. The bacteria uses this operon to convert its excess carbon source into polyhydroxybutyrate (PHB). R. eutropha stores PHB as an energy source (source). The  PhaC, PhaA, and PhaB gene expression leads to formation of pha synthase, acetoacetyl-CoA reductase, and 3-ketothiolase (source). These enzymes play role in converting acetyl-CoA into acetoactyl-CoA, which is converted to (R)-3-hydroxybutyryl-CoA. Finally, this product is converted to PHB (source).
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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) . R. eutropha 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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     The naturally existing operon PhaCAB in R. eutropha is known to produce PHB. However, literature has shown that the rearrangement of the operon to PhaCBA results in higher production of PHB because PhaB results in …(add more info from paper-> talk about molecular weight as well) (source). Hence, we decided to change the gene order from PhaCAB to PhaCBA. The operon rearrangement will lead to relatively higher expression of PhaB. We know from literature that PhaB is important for (add more info from paper and Imperial’s modelling…) (source). In addition to rearranging the gene order, the operon sequence was codon optimized to function in E. coli.  
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     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 <i>et. al</i>, 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 <i>et. al</i> 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.
 
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<p><center><img src="https://static.igem.org/mediawiki/2017/5/55/Calgary2017_GlycolysisCABConstruct.png" alt="Glycolysis CAB Construct" style="width:100%"></center></p>
 
<p><center><img src="https://static.igem.org/mediawiki/2017/5/55/Calgary2017_GlycolysisCABConstruct.png" alt="Glycolysis CAB Construct" style="width:100%"></center></p>
  
 
<p><center><img src="https://static.igem.org/mediawiki/2017/b/b4/Calgary2017_GlycolysisConstruct.png" alt="Glycolysis CBA Construct" style="width:100%"></center></p>
 
<p><center><img src="https://static.igem.org/mediawiki/2017/b/b4/Calgary2017_GlycolysisConstruct.png" alt="Glycolysis CBA Construct" style="width:100%"></center></p>
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<center><b>Figure 1.</b> Naturally existing phaCAB operon in <i>R. eutropha</i> on top and rearranged operon phaCBA on bottom.</center>
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<h2>Media/Culture composition & conditions </h2>
 
<h2>Media/Culture composition & conditions </h2>
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In order to test our gene construct, the operon was inserted into pET29(b)+. Competent <i>E. coli</i> (BL21) was then transformed with the plasmid. The overnights of successfully transformed cells was then used for our <a src="https://2017.igem.org/Team:Calgary/Experiments">experiments</a>. The different media composition used were glucose only and fermented synthetic feces supernatant (which is referred to as "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.</p>
  
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<h2>Results</h2>
 
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(rationale goes here)
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The results of the experiments for our phaCBA construct is given on <a src="https://2017.igem.org/Team:Calgary/Results"> results page</a> and <a src="http://parts.igem.org/Part:BBa_K2260000">our parts registry.</a>
 
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<h2>References</h2>
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Results. …
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<p>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.</p>
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(link to parts characterization)
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Revision as of 03:25, 31 October 2017

Header

Glycolysis

Glycolysis Pathway

Aim

In order to utilize acetyl-coA, which is a product downstream of the glycolysis pathway, a naturally occurring operon in R. eutropha 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) . R. eutropha stores PHB as an energy source (Hiroe et. al, 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 et. al, 2012).

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 & conditions

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 synthetic feces supernatant (which is referred to as "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.