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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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| − | == 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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| − | 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 consume an extra amount of higher fatty acids is achieved by overexpressing fadD gene.
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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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FadD Fatty Acyl-CoA Synthetase (Ligase) from E. coli. Contains a 8-Histidine tag immediately before the stop codon to allow gel identification and extraction. The FadD Fatty Acyl-CoA Synthetase (Ligase) converts fatty acids into fatty acyl-CoA using ATP.
