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 sTF05 were tested by measuring the fluorescence of an A. niger carring sTF05 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 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 did not. It should be noted that no growth was observed on the plates containing 0.6 g/L furfural (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]