Part:BBa_K3763003

pFadD promoter with LacI repressor regulating downstream GFP with RBS BBa_K3763000

pFadD_Lac is a fatty acid-sensitive promoter, one of the regulators in the enzymes of fatty acid biosynthesis in E. coli. It is composed of two fadR recognition sites. In the absence of fatty acids, an intrinsic transcription factor FadR acting as the repressor in the fatty acid metabolism system in E. coli. will attach to the promoter, and block the downstream gene expression. This has been proved to be effective by a previous iGEM team（NTHU_Taiwan）. [1][2] 2019 NTHU_Taiwan further modified pFadD promoter by replacing its CRP binding site with a LacI repressor binding site, which reduced expression leakage.

Figure 1. pFadD_Lac promoter and fadR.

We combine the RBS(B0030) with the promoter pFadD_Lac which is sensitive to fatty-acids and use GFP to test the performance of the promotor by measuring the fluorescence intensity.

Figure 2. The change in sequence.

Moreover, according to the fact that most RBS with higher binding efficiency had the repetitive sequence of "AGGG", or "AAA" after the start base, we improved a new part of RBS and tried to compare it with the original RBS (BBa_B0030) to see which is better in some specific occasions.

After the successful transformation of the plasmid, we used different concentrations of fatty acids for induction for different times. The expression level of eGFP was determined by fluorescence detection with a microplate reader. The results are as follows:

Figure 3. BBa_K3763002(pFadD_Lac-RBS BBa_B0030-GFP-rrnB T1 and T2 terminator, pDSW208-99-2) contains RBS without modification.

Figure 4. BBa_K3763003(pFadD_Lac-RBS BBa_K3763000-GFP-rrnB T1 and T2 terminator, pDSW208-99-2) Contains RBS with modification

According to the results, we can find that the performance of our fatty acids-sensitive promoter is improved to a certain extent. Taking 0.125× fatty acid concentration as an example, the fluorescence intensity of BBa_K3763003 is over 11000 after 6 hours, while the promoter of BBa_K3763002 is less than 10000 at three fatty acid concentrations. However, we can see that the optimized promoter still has a high expression leakage and low expression in a short time. However, the experimental results have been able to prove the function of the fatty acids-sensitive promoter, and partially prove the effectiveness and rationality of our design.

Reference
[1] https://2019.igem.org/Team:NTHU_Taiwan/Results
[2] http://2012.igem.org/Team:NTU-Taida#

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
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 938