Part:BBa_K3040015

pFadD promoter with one fadR binding sites from pFadBA 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. Yet, we further modified pFadD promoter by replacing its CRP binding site with a pLac promoter, which makes this promoter fully activated only when both the fatty acids and IPTG are present.

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

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 33
  Illegal XbaI site found at 219
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 33
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 33
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 33
  Illegal XbaI site found at 219
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 33
  Illegal XbaI site found at 219
  Illegal AgeI site found at 839
  Illegal AgeI site found at 951
  
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

1. FENG, Youjun; CRONAN, John E. Crosstalk of Escherichia coli FadR with global regulators in an expression of fatty acid transport genes. PloS one, 2012, 7.9: e46275.

2. ZHANG, Fuzhong; CAROTHERS, James M.; KEASLING, Jay D. Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nature biotechnology, 2012, 30.4: 354.