Difference between revisions of "Part:BBa K654059"

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Fatty Acyl-CoA Synthetase (Ligase) (fadD from E. coli) with 8-His Tag

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.

Fatty Acyl-CoA Synthetase is used in conjunction with Thioesterase to greatly increase the concentration of Fatty Acyl-CoA present in the cell, to increase the amount of bioproducts produced. It is used to produce Fatty Alcohols and Wax Esters in the 2011 Utah_State iGEM project.

Sequence and Features

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]

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 consume an extra amount of higher fatty acids is achieved by overexpressing fadD gene.

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