Difference between revisions of "Part:BBa K2260000"
Line 4: Line 4:
  
 
<h2>Overview</h2>
 
<h2>Overview</h2>
 +
 +
<table>
 +
 +
<tr>
 +
<th><b>Biobrick</b>: BBa_K2260000</th>
 +
                <td rowspan=4>      *****Insert Image****** </td>
 +
</tr>
 +
<tr>
 +
<th><b>Part type</b>: Composite</th>
 +
</tr>
 +
<tr>
 +
<th><b>Description</b>: PhaCBA operon w/ RBS</th>
 +
</tr>
 +
<tr>
 +
<th width=40%><b>Source</b>: <i>Ralstonia eutropha H16</i></th>
 +
</tr>
 +
</table>
  
 
<p style="text-indent: 4em;">
 
<p style="text-indent: 4em;">

Revision as of 04:24, 29 October 2017


PhaCBA operon w/ Histidine tags

Overview

Biobrick: BBa_K2260000 *****Insert Image******
Part type: Composite
Description: PhaCBA operon w/ RBS
Source: Ralstonia eutropha H16

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 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
<img src="HPLC_CBA_High.png"> <img src="HPLC_CBA_Low.png"> <img src="HPLC_CBA_Arbitrary.png">

Nile red suspension for confirmation 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]