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 FunsTF05 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 genome 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.

Figure 2: This is the figure text.

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]