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, in that it has a nuclear localization signal (NLS) and is codon optimised to Aspergillus niger. FunsTF18 is a fusion protein consisting of the DNA-binding domain from LexA, the ligand sensing domain from 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)). 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 FunsTF18 carry mutant 12 of HbaR, which had the following mutations: A45S, L64I, F85M, A86G, A88Y and Y96S.

The transactivation domain B112 is from E. coli, and it 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 of the protein to the nucleus (Garcia-Bustos et. al (1991)).

Characterization

The functionality of FunsTF18 was tested by measuring the fluorescence of A. niger expression FunsTF18 and the mCherry reporter. The A. niger is grown on solid media plates and these 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 expression 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 functional.

Figure 1: Pictures of fluorescent A. niger, carrying 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.

In addition, to characterising FunsTF18 in A. niger grown on solid media, the A. niger that expressed FunsTF18 was tested in liquid media. The fluorescence is measured at four time points and they are 2 hours (Figure 4A), 26 hours (Figure 4B), 47 hours (Figure 4C), and 70 hours (Figure 4D) after induction. The variance of the fluorescence at the four time points are minimal. Genomically integrated BBa_K3046004 has the highest fluorescence and FunsTF18 displays fluorescence slightly above BBa_K4129025 at all time points (Figure 4). Inducibility was not observed in liqiud either (figure 4).

Figure 2:Barplot of log transformed fluorescence (RFU) with standard deviation displayed as arrows. The fluorescence was from A. niger carrying genomically integrated BBa_K3046004, BBa_K4129025 or FunsTF18. The A. niger was grown in minimal media and it was uninduced (blue) or induced with 0.6 g/L furfural (red) or 2 mM benzoic acid (green). The fluorescence was measured A) 2 hours, B) 26 hours, C) 47 hours, and D) 70 hours after induction.

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]