Part:BBa_K4129101

The fungal synthetic transcription factor, FunsTF02 (LexA-SL-HbaR-B112-SV40)

FunsTF02 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)). FunsTF02 deviates from sBAD, because it has an nuclear localization signal (NLS) and is codon optimised to A. niger. FunsTF02 is a fusion protein consisting of the DNA-binding domain LexA, the ligand sensing domain HbaR, transactivation domain B112 and the nuclear localization signal (NLS) SV40.

The designed function of FunsTF02 is to be used as a transcription factor that can initiate transcription from the 6xLexO minimal promoter (BBa_K4129115). This sTF can be the sensing part of a biosensor.

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 FunsTF02. 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)).

The transactivation domain B112 is from E. coli, which was 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 og the protein to the nucleus (Garcia-Bustos et. al (1991)).

Characterization

The functionally of FunsTF02 was tested by measuring the fluorescence of an A. niger carring FunsTF05 and the mCherry reporter (BBa_K4129123). The A. niger is grown on solid media plates. The plates contained either minimal media, minimal media with mM benzoic acid or MM with 0.6 g/L furfural.

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. It is observed that genomically integrated BBa_K3046004 displays fluorescence and the negative control of BBa_K4129025 did not (figure 1).

Figure 1: Pictures of fluorescent A. niger, which carries either BBa_K4129025 or genomically integrated BBa_K3046004. The picture are taken with 1.04 seconds exposure time. The A. niger is grown on plates containing minimal media (MM), MM with 2 mM benzoic acid or MM with 0.6 g/L furfural. The fluorescent is depicted as grey-white intensity.

Fluorescence from FunsTF02 was only obsvered when the A. niger was grown minimal media. The fluorescence is localised to the perimeter of the colony and the with a low intensity (figure 2). FunsTF04 is funtional.

Figure 2: Pictures of fluorescent A. niger, which carries either FunsTF02 or FunsTF57. The picture are taken with 1.04 seconds exposure time. The A. niger is grown on plates containing minimal media (MM), MM with 2 mM benzoic acid or MM with 0.6 g/L furfural. 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 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]