Part:BBa_K3664006:Experience
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
Applications of BBa_K3664006
User Reviews
UNIQ99b05eca75132346-partinfo-00000000-QINU
UNIQ99b05eca75132346-partinfo-00000001-QINU
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.