Difference between revisions of "Part:BBa K2260000"
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| − | The naturally occuring phaCAB operon in <i>R. eutropha </i> H16 is involved in biosynthesis of poly[(R)-3-hydroxybutyrate] (PHB) ___!!source!!____. It utilizes | + | The naturally occuring phaCAB operon in <i>R. eutropha </i> H16 is involved in biosynthesis of poly[(R)-3-hydroxybutyrate] (PHB) ___!!source!!____. It utilizes acetyl-coA, which is a product of the glycolysis pathway ___!!source!!____. 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 ___!!source!!____. </p> |
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PHB produced from our construct was verified using crotonic acid digest __!!link to protocol!!__. The HPLC results verified that the white powder obtained from extraction of PHB from bacteria was indeed PHB. The following table shows the HPLC results: | PHB produced from our construct was verified using crotonic acid digest __!!link to protocol!!__. The HPLC results verified that the white powder obtained from extraction of PHB from bacteria was indeed PHB. The following table shows the HPLC results: | ||
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<h2>Nile red suspension for confirmation of PHB</h2> | <h2>Nile red suspension for confirmation of PHB</h2> | ||
Revision as of 03:06, 30 October 2017
PhaCBA operon w/ Histidine tags
Overview
The naturally occuring phaCAB operon in R. eutropha H16 is involved in biosynthesis of poly[(R)-3-hydroxybutyrate] (PHB) ___!!source!!____. It utilizes acetyl-coA, which is a product of the glycolysis pathway ___!!source!!____. 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 ___!!source!!____.
In order to utilize acetic acid present in fermented human feces ___!!source!!____, we decided to incorporate the phaCAB operon. However, literature has shown that the rearrangement of operon to phaCBA leads to higher amount of production of PHB ___!!source!!____. Thus, we obtained the operon from <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1149051">BBa_K1149051</a> and rearranged the construct from phaCAB to phaCBA and added histidine tags. The iGEM suffix is at the end of the gene construct.
PHB Weights from fermented "syn poo" supernatant ___!!source!!____
HPLC of PHB
PHB produced from our construct was verified using crotonic acid digest __!!link to protocol!!__. The HPLC results verified that the white powder obtained from extraction of PHB from bacteria was indeed PHB. The following table shows the HPLC results:
| High | Low | Arbitrary |
|---|---|---|
| High | Low | Arbitrary |
 |
 |
 |
Nile red suspension for confirmation of PHB
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]