Part:BBa_K3040014

pFadD promoter with pLac regulating downstream RFP

Background

FadD promoter is one of the regulator in the enzymes of fatty acid biosynthesis in E. coli. It is composed of two fadR recognition sites (The sequence is slightly different from the fadR recognition site in fadBA promoter), one ArcA binding site and one CRP binding site.

LacI repressor and its operator together form a genetic switch, the lac operon . The switch functions when inducer molecules alter the conformation of the repressor in a specific manner. In the presence of a particular metabolite such as lactose, the repressor undergoes a conformational change that reduces its affinity for the operator.

Figure1. The fadD promoters of E. coli.

Mechanism and Design

Having its own different sequence of fadR binding site makes pFadD promoter having different strength with pFadBA. Based on this fact, we tried pFadD promoter to take place of the weak pFadBA promoter. Moreover, with two fadR recognition sites and various repressor binding sites, native in pFadD, we assume a better result with the lower leakage in our pFadD promoter.

Moreover, we further modified pFadD promoter by replacing its CRP binding site with a LacI repressor binding site, which makes the rfp expression was tightly repressed in the absence of IPTG. Therefore, the hybrid promoter can be fully activated only when both the fatty acids( in our case, we add oleic acid) and IPTG are present. By doing so, we expect to achieve the lower leakage than the natural acyl-CoA responsive promoter pfadBA submitted by iGEM12_NTU-Taida (BBa_K817002).

Expression in E. coli

The fadD-lac promoter was used to transform E. coli DH5α.

Result

We can see clearly that pFadD_Lac, pFadD 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 also has reduction in leakage, we assumed them not functioning since they show no changes in expression as the concentration of fatty acid rise.

Figure 5. Protein expression of fatty acid promoter pFadD, pFadD-FadR, pFadD–lac (n=3).

Figure 6. 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 prefix found in sequence at 30
  Illegal suffix found in sequence at 230
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 30
  Illegal SpeI site found at 231
  Illegal PstI site found at 245
  Illegal NotI site found at 36
  Illegal NotI site found at 238
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 30
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 30
  Illegal suffix found in sequence at 231
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 30
  Illegal XbaI site found at 45
  Illegal SpeI site found at 231
  Illegal PstI site found at 245
  Illegal AgeI site found at 863
  Illegal AgeI site found at 975
  
• 1000
  COMPATIBLE WITH RFC[1000]