Part:BBa_K4129114

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

FunsTF70 is a synthetic transcription factor (sTF). FunsTF05 should function as a transcription factor that can initiate transcription of the 6xLexO minimal promoter (BBa_K4129115). This sTF will be the sensing part of the biosensor.

FunsTF70 is a fusion protein consisting of the DNA-binding domain: lexA, ligand sensing domain: HbaR16, transactivation domain; VP16 and the nuclear localization signal (NLS) SV40. The linker between LexA and HbaR16 was a longer version (Ottoz et. al (2014) compared to sBAD (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)), and it is the DNA binding domain that interacts with LexO that is used in FunsTF70. HbaR is a transcriptional factor from Rhodopseudomonas palustris that initiates transcription in the presence of benzoic acid or in the presence of benzoic acid derivatives (Egland. et al (2000) ,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.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 sTF70 and the mCherry reporter. 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 was assessed using the Vilber Fusion FX imager system to measure fluorescence shown as grey-white intensity. The exposure time was normalised to the fluorescences from genomic integrated BBa_K3046004, and this enabled comparison between plates. The exposure time used was 0.72 seconds. It is observed that genomic integrated BBa_K3046004 displays fluorescence and the negative control of BBa_K4129025 is barely visible (figure 1).

Figure 1: Pictures of fluorescent A. niger, which carries either genome integrated BBa_K3046004, BBa_K4129025 or FunsTF70. The picture are taken with 0.72 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
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 945
  Illegal BamHI site found at 607
  Illegal XhoI site found at 800
  Illegal XhoI site found at 1237
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]