Part:BBa_K3040012

Two FadR binding sites with pLac promoter regulating downstream RFP

We create a hybrid Fatty acid/acyl-CoA-regulated promoter by the combination of pLac with a modified promoter pFadBA. It is pointed out by the previous iGEM team, UPF CRG Barcelona_2018 iGEM, that the native promoter pFadBA has great leakage and is a weak promoter that isn’t ideal enough for our system. Therefore, to enhance the sensitivity and reduce the leakage of the promoter pFadBA for making it more suitable for our system, we improved our promoter based on the previous pfadBA DNA sequence from NTU_Taida 2012. pFL2 promoter has two fadR recognition sites, one between its -10 and -35 region and the other lies just before the -35 region, with a promoter pLac behind -10 region. pFL2 promoter is designed to respond to changes of both fatty acid and an exogenous inducer, IPTG. The hybrid promoter pFL2 is fully activated only when both the fatty acids and IPTG are present.

Result

As we can see from fig.2, the expression of PFL1 (BBa_K3040011) was greatly enhanced to 2 folds under IPTG induction. Yet, there is no difference between the inductions of IPTG in PFL2 (BBa_K3040012). Therefore, PFL1 is proved to successfully express while PFL2 fails to function.
Here, we conjecture that two fadR binding sites might have been over repressing the expression of the promoter. Higher fatty acid induction might help, yet, the solubility of oil has limited us for further experiments.

Figure 1. Relative protein expression of fatty acid pFadBA-Lac-1 after 16 hours of induction under different fatty acid concentration (n=3).

Figure 2. IPTG induction effects on protein expression of fatty acid pFadBA-Lac-2 (PFL2) after 16 hours of induction under 5mM fatty acid (n=3).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 755
  Illegal AgeI site found at 867
  
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

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.