Part:BBa_K4129114

The fungal synthetic transcription factor, FunsTF70 (LexA-LL-HbaR16-VP16-SV40)

FunsTF70 is a synthetic transcription factor (sTF). FunsTF70 should function as a transcription factor that can initiate transcription of the 6xLexO minimal promoter (BBa_K4129115). This sTF is designed to be the sensing part of the biosensor.

FunsTF70 is a fusion protein consisting of the DNA-binding domain from LexA, the ligand sensing domain from HbaR16, the transactivation domain from VP16 and the nuclear localization signal (NLS) SV40. The linker between LexA and HbaR16 was a longer linker (Ottoz et. al (2014) compared to the linker used in sBAD, which was the reference sTF (Castaño-Cerezo et. al (2020)).

LexA is a repressor that regulates the SOS response in E. coli (Radman. 1975). LexA binds to a specific DNA motif, LexO (Erill. et al (2003)). HbaR is a transcription factor from Rhodopseudomonas palustris that initiates transcription in the presence of benzoic acid (Egland. et al (2000)) or in the presence of benzoic acid derivatives (Castaño-Cerezo et. al (2020)). We created 16 mutants of HbaR and FunsTF70 carried mutant 16 of HbaR, which had the following mutations: L64I, F85H, A86G, A90Y and L97G.

Viral Protein 16 (VP16) from Herpes simplex virus type 1 is a transcription factor that uses a transactivation domain to recruit the RNA polymerase II (Hirai et al. (2010)).The NLS SV40 is a small peptide sequence of PKKKRKV that enables transport of the protein to the nucleus (Garcia-Bustos et. al (1991)).

Characterization

The functionality of sTF70 was tested by measuring the fluorescence of A. niger expression FunsTF70 and the mCherry reporter. The A. niger is grown on solid media plates and the plates contained either minimal media or minimal media with 2 mM benzoic acid.

The fluorescence of the plates was assessed, after four days of incubation at 30℃, using the Vilber Fusion FX imager system. The intensity of the fluorescences was presented as grey-white. The exposure time was normalised to the fluorescences from genomic integrated BBa_K3046004. The exposure time was set to 0.72 seconds. It is observed that genomic integrated BBa_K3046004 displays fluorescence and the negative control of BBa_K4129025 is barely visible. The fluorescence from mCherry was clear on both plates when funsTF70 was expressed. This indicates that FunsTF70 functions as a transcription factor, that promotes constitutive expression. The expression level of mCherry was not to the same expression level as a strong constitutive promoter like BBa_K3046004 (figure 1).

Figure 1: Pictures of fluorescent A. niger, which carrying either genome integrated BBa_K3046004, BBa_K4129025 or FunsTF70. The pictures are taken with 0.72 seconds exposure time. The A. niger is grown on plates containing minimal media (MM) or MM with 2 mM benzoic acid. The fluorescent is depicted as grey-white intensity.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 673
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 860
  Illegal BamHI site found at 607
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 765
  
• 1000
  COMPATIBLE WITH RFC[1000]