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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<p><p>In many species of bacteria, acetyl-CoA results from the breakdown of glucose through glycolysis and the breakdown of fatty acids through beta-oxidation. 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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<p>Hiroe <i>et al.</i> showed that the content of PHB is dependent on the expression of <i>phaB</i>. This rearrangement from <i>phaCAB</i> to <i>phaCBA</i> will lead to higher expression of <i>phaB</i> compared to that of the native operon, resulting in more PHB produced. However, the molecular weight of PHB was not affected by the different expression of <i>phaB</i>, indicating the chemical structure of the polymer remained consistent.</p>
 
<p>Hiroe <i>et al.</i> showed that the content of PHB is dependent on the expression of <i>phaB</i>. This rearrangement from <i>phaCAB</i> to <i>phaCBA</i> will lead to higher expression of <i>phaB</i> compared to that of the native operon, resulting in more PHB produced. However, the molecular weight of PHB was not affected by the different expression of <i>phaB</i>, indicating the chemical structure of the polymer remained consistent.</p>
  
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<h2>Media/Culture composition</h2>
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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 <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 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>Media Composition</h2>
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<p>The <i>phaCBA</i> operon can acetyl-CoA produced from the breakdown of glucose and volatile fatty acids (VFAs) 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)+ under the control of an IPTG-inducible promoter and transformed into <i>E. coli</i> BL21(DE3). Various media were used to test the ability of the recombinant bacteria to use glucose and VFAss to produce PHB.  Differing combinations of these initial substrates were compared to demonstrate our operon’s working efficiency.</p>
  
 
<h2>Results</h2>
 
<h2>Results</h2>

Revision as of 04:34, 31 October 2017

Header

Glycolysis

Glycolysis Pathway


Aim

In many species of bacteria, acetyl-CoA results from the breakdown of glucose through glycolysis and the breakdown of fatty acids through beta-oxidation. 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

In R. eutropha, the aforementioned genes exist in the order phaCAB. However, literature has shown that the rearrangement of the operon to phaCBA results in higher production of PHB (Hiroe et al., 2012). Therefore, we decided to change the order of our operon.


Glycolysis CAB Construct


Glycolysis CBA Construct

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

Hiroe et al. showed that the content of PHB is dependent on the expression of phaB. This rearrangement from phaCAB to phaCBA will lead to higher expression of phaB compared to that of the native operon, resulting in more PHB produced. However, the molecular weight of PHB was not affected by the different expression of phaB, indicating the chemical structure of the polymer remained consistent.


Media Composition

The phaCBA operon can acetyl-CoA produced from the breakdown of glucose and volatile fatty acids (VFAs) 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)+ under the control of an IPTG-inducible promoter and transformed into E. coli BL21(DE3). Various media were used to test the ability of the recombinant bacteria to use glucose and VFAss to produce PHB. Differing combinations of these initial substrates were compared to demonstrate our operon’s working efficiency.

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.