Part:BBa_K3664006
ACS2+WS2->gene sequence
The gene sequence contains two target genes: ACS2 (long chain acyl CoA synthase) and WS2 (wax ester synthase).It could express two proteins, CoA ligase and acyltransferase.The function of the two proteins as follows. CoA ligase:R1-COOH+CoA=R1-CO-S-CoA; acyltransferase:R1-CO-S-CoA+R2-OH=R1-COOR2 At the same time, in our design, we used homotail enzyme to splice two segments of genes (XbaI and NheI), and contained a terminator of acs2 and a promoter of WS2 between the two segments (ACS2 / WS2)
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1549
Illegal EcoRI site found at 1597
Illegal XbaI site found at 1739
Illegal PstI site found at 199
Illegal PstI site found at 637
Illegal PstI site found at 889
Illegal PstI site found at 2476
Illegal PstI site found at 2875
Illegal PstI site found at 2917
Illegal PstI site found at 2980 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1549
Illegal EcoRI site found at 1597
Illegal PstI site found at 199
Illegal PstI site found at 637
Illegal PstI site found at 889
Illegal PstI site found at 2476
Illegal PstI site found at 2875
Illegal PstI site found at 2917
Illegal PstI site found at 2980 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1549
Illegal EcoRI site found at 1597 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1549
Illegal EcoRI site found at 1597
Illegal XbaI site found at 1739
Illegal PstI site found at 199
Illegal PstI site found at 637
Illegal PstI site found at 889
Illegal PstI site found at 2476
Illegal PstI site found at 2875
Illegal PstI site found at 2917
Illegal PstI site found at 2980 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1549
Illegal EcoRI site found at 1597
Illegal XbaI site found at 1739
Illegal PstI site found at 199
Illegal PstI site found at 637
Illegal PstI site found at 889
Illegal PstI site found at 2476
Illegal PstI site found at 2875
Illegal PstI site found at 2917
Illegal PstI site found at 2980
Illegal NgoMIV site found at 564
Illegal AgeI site found at 430
Illegal AgeI site found at 646
Illegal AgeI site found at 658
Illegal AgeI site found at 1051
Illegal AgeI site found at 1312
Illegal AgeI site found at 2569
Illegal AgeI site found at 3178 - 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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