Part:BBa_K3040013
Native pFadD promoter regulating downstream RFP
pFadD promoter is one of the regulators in the enzymes of fatty acid biosynthesis in E. coli. It is composed of two fadR recognition sites, and one CRP binding site. The fatty acid metabolism system of E. coli is consist of many parts, fadD, fadL, fadR, fadA, etc. Interestingly, each enzyme in this family has its own different sequence of fadR recognition site in its promoter, which makes these promoter having different strength, yet can still be interchangeable for us to apply in our system. Based on this fact, we tried pFadD promoter to take place of the weak pFadBA promoter. With two fadR recognition sites native in pFadD, we assume a better result in the sensitivity and a lower leakage in our pFadD promoter.
Result
We can see clearly that pFadD_Lac (BBa_K3040014), pFadD (BBa_K3040013) promoter with an additional lac binding site, has relatively low leakage and has 2-3 fold increase in expression as the fatty acid concentration rises. Though compared to the original promoter pFadBA, both pFadD and pFadD_FadR (BBa_K3040015) also has a reduction in leakage, we assumed them not functioning since they show no changes in expression as the concentration of fatty acid rise.
Figure 1. Protein expression of fatty acid promoter pFadD, pFadD-FadR, pFadD–lac after 16 hours of induction under 5mM fatty acid (n=3).
Figure 2. Protein expression of fatty acid promoter pFadD, pFadD-FadR, pFadD–lac after 16 hours of induction under different fatty acid concentration (n=3).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 755
Illegal AgeI site found at 867 - 1000COMPATIBLE WITH RFC[1000]
Reference
FENG, Youjun; CRONAN, John E. Crosstalk of Escherichia coli FadR with global regulators in the expression of fatty acid transport genes. PloS one, 2012, 7.9: e46275.
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