Part:BBa_K861020
FadE ,acyl-CoA dehydrogenase
The first step in the beta-oxidation pathway involves the conversion of acyl-CoA into enoyl-CoA. This process is catalyzed by acyl-CoA dehydrogenase FadE. Its action results in the introduction of a trans double-bond between C2 and C3 of the acyl-CoA thioester substrate.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 584
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 367
Illegal SapI.rc site found at 1240
Illegal SapI.rc site found at 2383
Characterized by BNU-China 2019
In order to have our engineered microbe consume the extra in-taken fat, we overexpress fadE gene derived from E. coli K-12 DH5alpha genome in our engineered intestinal microbe to catalyze dehydrogenation process from fatty acyl-CoA to fatty enoyl-CoA in fatty acid beta oxidation.
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 an increase in fatty acids consumption upon induction. As is shown in Fig. 1, the experimental group consumes nearly 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 fadE 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 584
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 367
Illegal SapI.rc site found at 1240
Illegal SapI.rc site found at 2383
| n/a | FadE ,acyl-CoA dehydrogenase |