Part:BBa_K4129106

The fungal synthetic transcription factor, FunsTF18 (LexA-SL-HbaR12-B112-SV40)

FunsTF18 is a synthetic transcription factor (sTF) based on sensor of benzoic acid derivatives (sBAD), which is a sTF in S. cerevisiae (Castaño-Cerezo et. al (2020)). FunsTF18 differ from sBAD, because it has an nuclear localization signal (NLS) and is codon optimised to A. niger. FunsTF18 is a fusion protein consisting of the DNA-binding domain LexA, the ligand sensing domain HbaR12, transactivation domain B112 and the nuclear localization signal (NLS) SV40.

LexA is a repressor that regulates the SOS response in E. coli (Radman. 1975). LexA binds to a specific DNA motif, namely LexO sites (Erill. et al (2003)), and it is the DNA binding domain interacting with LexO that is used in FunsTF16. 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 FunsTF16 carried mutant 12 of HbaR, which had the following mutations: A45S, L64I, F85M, A86G, A88Y and Y96S.

The transactivation domain B112 is from E. coli, which were experimentally proven to initiate transcription of a synthetic promoter in S. cerevisiae (Ottoz et. al (2014)). 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 functionally of sTF70 were tested by measuring the fluorescence of an A. niger carring FunsTF18 and the mCherry reporter. The A. niger is grown on solid media plates. The plates contained either minimal media or minimal media with 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 genomically integrated BBa_K3046004 and the exposure time was set to 0.72 seconds. It is observed that genomically integrated BBa_K3046004 displays fluorescence and the negative control of BBa_K4129025 is barely visible. The A. niger carring FunsTF18 is visble, which indicates more fluorescence than BBa_K4129025. In additio, FunsTF18 has fluorescences with and without 2 mM benzoic acid in the media, but far from expression BBa_K3046004 (figure 1). In conclusion FunsTF18 is also functional.

Figure 1: Pictures of fluorescent A. niger, which carries either genome integrated BBa_K3046004, BBa_K4129025 or FunsTF18. The picture 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 622
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 809
  Illegal BamHI site found at 1148
  Illegal XhoI site found at 1297
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 714
  
• 1000
  COMPATIBLE WITH RFC[1000]