Part:BBa_K3763001

Over-expression of FadR

Overview

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.

Figure 1. pFadD_Lac promoter and fadR.

To better reduce expression leakage, we overexpressed FadR, which has been shown effective to increase bacterial sensitivity to fatty acids.

Characterization

We cloned genomic DNA FadR from E.coli MG1655 and digested it with HindIII and XbaI. Plasmid (pBAD33)was extracted using an extraction kit, and digested with HindIII and XbaI as well. Right bands of pBAD33 vector (around 5000 bp) and FadR gene sequence (1038bp) were observed.

Figure 2.Enzyme digestion of plasmids and gene we need (pBAD33 and FadR gene). L1, L2: FadR gene digested with HindIII and XbaI; L4, L5: pBAD33 digested with HindIII and XbaI.

The gene sequencing results indicated that we successfully get FadR.

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

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961