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 actyl-coA, which is a product of the glycolysis pathway ___!!source!!____.  
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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 actyl-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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This part contains the naturally occurring phaC, phaA, and phaB operon rearranged to phaC, phaB, and phaA. It also contains his tags and ribosome binding sites (RBS). The iGEM suffix is at the end of the gene construct. Transcription along the operon leads to expression of the enzymes: 3-ketothiolase, acetoacetyl-CoA reductase, and pha synthase. These enzymes are involved in the pathway that converts acetyl-CoA to polyhydroxybutyrate (PHB). The operon was rearranged based on results from literature <source>, which showed that the phaCBA operon leads to higher production of PHB compared to phaCAB.  
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This part can be used to utilize the end-product of glycolysis pathway i.e. acetyl-CoA. After a series of three reactions, PHB is obtained.
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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.
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This part was used in our project to utilize acetic acid, which is present in the supernatant collected from fermented synthetic feces. The part was tested using E. coli (BL21) as the chassis. The bacteria containing the vector was The amount of PHB produced from E. coli at known cell density was recorded using mass balance. Furthermore, HPLC and nile red staining was used to confirm the presence of PHB.
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<h2>PHB from fermented "syn poo" supernatant ___!!source!!____</h2>
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The part was tested using E. coli (BL21) as the chassis. The bacteria containing the vector was The amount of PHB produced from E. coli at known cell density was recorded using mass balance. Furthermore, HPLC and nile red staining was used to confirm the presence of PHB.
 
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Revision as of 06:33, 27 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 actyl-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 from fermented "syn poo" supernatant ___!!source!!____

The part was tested using E. coli (BL21) as the chassis. The bacteria containing the vector was The amount of PHB produced from E. coli at known cell density was recorded using mass balance. Furthermore, HPLC and nile red staining was used to confirm the presence of PHB.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]