Part:BBa_K3664010
ppfadD+WS/DGAT
This part contains two genes: ppfadD and WS / DGAT. Between the two genes, there is a terminator of ppfadd gene and a promoter of WS / DGAT gene. At the same time, these two parts are connected by the way of tail enzyme connection, which is homocaudal enzyme (XbaI and NheI).The two genes expressed acyl coenzyme A synthetase and wax ester synthase.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2101
Illegal XbaI site found at 1751
Illegal PstI site found at 196
Illegal PstI site found at 256
Illegal PstI site found at 634
Illegal PstI site found at 709
Illegal PstI site found at 2458
Illegal PstI site found at 2839 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2101
Illegal PstI site found at 196
Illegal PstI site found at 256
Illegal PstI site found at 634
Illegal PstI site found at 709
Illegal PstI site found at 2458
Illegal PstI site found at 2839 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2101
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2101
Illegal XbaI site found at 1751
Illegal PstI site found at 196
Illegal PstI site found at 256
Illegal PstI site found at 634
Illegal PstI site found at 709
Illegal PstI site found at 2458
Illegal PstI site found at 2839 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2101
Illegal XbaI site found at 1751
Illegal PstI site found at 196
Illegal PstI site found at 256
Illegal PstI site found at 634
Illegal PstI site found at 709
Illegal PstI site found at 2458
Illegal PstI site found at 2839
Illegal AgeI site found at 427
Illegal AgeI site found at 600
Illegal AgeI site found at 643
Illegal AgeI site found at 655
Illegal AgeI site found at 847
Illegal AgeI site found at 1069
Illegal AgeI site found at 1333 - 1000COMPATIBLE WITH RFC[1000]
Purpose of the experiment
To verify their functionality, we constructed two plasmids pET-28a (WS / PPF) and pET-28a (WS2 / ACS2) .In the verification experiment, we used hydroxyhexadecanoic acid as the substrate.
We expressed pet-28a-WS2 / ACS2 and pet-28a-WS / PPF into E.coli MG1655 At the same time, the related plasmids were also expressed in E.coli MG1655 (fade knockout to exclude the effect of β- oxidation).
Experimental results and analysis
1.SDS protein electrophoresis
Molecular weight of target protein
WS-DGAT 51.8 kDa
WS2 52.5 kDa
AcsII 61.9kDa
PPF 61.9kDa
Through SDS protein electrophoresis, we can find that by comparing with marker, we can confirm that our gene expression, E.coli has the enzyme we need.
2.Growth curve of E. coli
After the addition of foreign genes, the ability of substrate assimilation was enhanced, and the growth curve showed satisfactory results; after the fade gene was knocked out, it had a certain impact on the metabolism of bacteria, and the growth and metabolism slowed down, but it still showed that it could grow normally, indicating that the new pathway we constructed was feasible. After knockout, we could exclude the metabolism of fatty acids by the bacteria itself influence.
3.Results of GC-MS with hydroxyhexadecanoic acid as substrate
We found that the substrate content of E.coli MG1655 (fade knockout, including pet-28a-ws2 / acs2 and pet-28a-ws / PPF) was significantly reduced in E.coli MG1655 (fade knock-out, including pet-28a-WS2 / ACS2 and pet-28a-WS / PPF), which indicated that the fatty acid CoA ligase and acyltransferase metabolic pathways were successfully constructed.
4.GC-MS diagram of products
By GC-MS analysis, we detected a high content of cyclohexadecanolide, which indicated that our fatty acid CoA ligase and acyltransferase metabolic pathways were successfully constructed and reacted with the substrate successfully. Although the polymerization has taken place, it also proves the feasibility of our metabolic pathway to a certain extent. At the same time, it is also a question that we need to explore in the future.
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