Difference between revisions of "Part:BBa K861010"

 
 
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<partinfo>BBa_K861010 short</partinfo>
 
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In Escherichia coli, LCFAs are transported and activated as fatty acyl-CoAs .The process of acetylation is catalyzed by FadD, the bacterial acyl-CoA synthetase (ACS).To enter the cell, MCFAs and LCFAs must be activated by theacyl-CoA synthetase (fadD gene product). And it is repressed by FadR
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In E.coli, after fatty acids being transported into the cell, to be degraded, they must be activated by the acyl-CoA synthetase-- FadD to form CoA derivatives. Also, FadD is shown to be essential for fatty acids transportation.
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K861010 parameters</partinfo>
 
<partinfo>BBa_K861010 parameters</partinfo>
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== Characterized by BNU-China 2019 ==
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In order to have our engineered microbe consume the extra in-taken fat, we overexpress fadD gene derived from E. coli K-12 DH5alpha genome in our engineered intestinal microbe to promote degradation of higher fatty acids which would otherwise be assimilated by human body. The catalysate fatty acyl-CoA also enhances the general fatty acids degradation by relieving the overall inhibiting effect of regulatory factor fadR towards β-oxidation. [1]
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<div style="text-align:center">
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<table border="solid"  width="500px" height="150px" cellspacing="0" cellpadding="10" frame="solid" rules="solid" style="margin: auto">
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<tr align="center" valign="center" bgcolor="66CCFF" >
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<td colspan="2"><font size="3"><b>fadD</b></font></td>
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</tr>
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<tr >
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<td><font size="2"><b>Function</b></font></td>
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<td>Fatty Acyl-CoA Synthetase</td>
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</tr>
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<tr>
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<td><font size="2"><b>Use in</b></font></td>
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<td>Prokaryotes</td>
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</tr>
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<tr>
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<td><font size="2"><b>RFC standard</b></font></td>
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<td>RFC10 compatible</td>
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</tr>
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<tr>
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<td><font size="2"><b>Backbone</b></font></td>
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<td>pSB1C3</td>
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</tr>
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<tr>
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<td><font size="2"><b>Derived from</b></font></td>
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<td>Escherichia. coli DH5alpha </td>
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</tr>
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</table>
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</div>
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Considering that sodium oleate has a generally steady and relatively high content in most kinds of fat, we select it to test relative general consumption of higher fatty acids.
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We take E. coli introduced with a vector with the same backbone as control group. Compared to it, the experimental group shows a significant increase in fatty acids consumption upon induction. As is shown in Fig. 1, the experimental group consumes more than twice as much sodium oleate as the control group within 2 and 4 hours, indicating enhancement of β-oxidation consuming an extra amount of higher fatty acids is achieved by overexpressing fadD gene.
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[[Image:2019_BNU-China_BBa_K654059_pic1.png | border | center | 400px]]<br>
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<div class = "center">Figure 1 Consumption of sodium oleate</div>
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<font size="4"><b>Experimental approach</b></font>
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1.Transform the plasmids into E. coli DH5α competent cells.
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2.A strain containing a vector with same backbone is used as control. Experimental groups and control groups are cultured in LB-ampicillin (50 ng/µl) medium overnight before being diluted with equal amount of LB-ampicillin (50 ng/µl) medium containing 400 mM sodium oleate, making the final concentration of oleate 200 mM.
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3.Both groups are induced with 5 mM IPTG and sampled at 0 hr, 2 hr and 4 hr. Centrifuge samples and take the supernatant.
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4.Measure the fatty acids concentration through enzyme linked immunosorbent assay (Shuangying FFA ELISA kit).
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5.Calculate and compare the sodium oleate consumption of experimental group and control group.
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6.Three repicas are tested in each group.
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<font size="4"><b>Reference</b></font>
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[1] Zhang Han‐Xing. Screening of PoIyhydroxyalkanoates producing bacteria and its expression and metabolic mechanism in E. coli engineered bacteria: [D]. Jinan: Shandong University, 2006
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K654059 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K654059 parameters</partinfo>
 
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Latest revision as of 09:41, 16 October 2019

FadD, an inner membrane-associated acyl-CoA synthase

In E.coli, after fatty acids being transported into the cell, to be degraded, they must be activated by the acyl-CoA synthetase-- FadD to form CoA derivatives. Also, FadD is shown to be essential for fatty acids transportation.

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]


Characterized by BNU-China 2019

In order to have our engineered microbe consume the extra in-taken fat, we overexpress fadD gene derived from E. coli K-12 DH5alpha genome in our engineered intestinal microbe to promote degradation of higher fatty acids which would otherwise be assimilated by human body. The catalysate fatty acyl-CoA also enhances the general fatty acids degradation by relieving the overall inhibiting effect of regulatory factor fadR towards β-oxidation. [1]

fadD
Function Fatty Acyl-CoA Synthetase
Use in Prokaryotes
RFC standard RFC10 compatible
Backbone pSB1C3
Derived from Escherichia. coli DH5alpha

Considering that sodium oleate has a generally steady and relatively high content in most kinds of fat, we select it to test relative general consumption of higher fatty acids.

We take E. coli introduced with a vector with the same backbone as control group. Compared to it, the experimental group shows a significant increase in fatty acids consumption upon induction. As is shown in Fig. 1, the experimental group consumes more than twice as much sodium oleate as the control group within 2 and 4 hours, indicating enhancement of β-oxidation consuming an extra amount of higher fatty acids is achieved by overexpressing fadD gene.


2019 BNU-China BBa K654059 pic1.png

Figure 1 Consumption of sodium oleate

Experimental approach

1.Transform the plasmids into E. coli DH5α competent cells.

2.A strain containing a vector with same backbone is used as control. Experimental groups and control groups are cultured in LB-ampicillin (50 ng/µl) medium overnight before being diluted with equal amount of LB-ampicillin (50 ng/µl) medium containing 400 mM sodium oleate, making the final concentration of oleate 200 mM.

3.Both groups are induced with 5 mM IPTG and sampled at 0 hr, 2 hr and 4 hr. Centrifuge samples and take the supernatant.

4.Measure the fatty acids concentration through enzyme linked immunosorbent assay (Shuangying FFA ELISA kit).

5.Calculate and compare the sodium oleate consumption of experimental group and control group.

6.Three repicas are tested in each group.


Reference

[1] Zhang Han‐Xing. Screening of PoIyhydroxyalkanoates producing bacteria and its expression and metabolic mechanism in E. coli engineered bacteria: [D]. Jinan: Shandong University, 2006

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]