Part:BBa_K4129102

The fungal synthetic transcription factor, FunsTF04 (LexA-LL-Hmox1-B112-SV40)

FunsTF04 is a synthetic transcription factor (sTF). FunsTF04 is designed to function as a transcription factor that can initiate transcription from the 6xLexO minimal promoter (BBa_K4129115). This sTF is designed to be the sensing part of a biosensor.

FunsTF04 is a fusion protein consisting of the DNA-binding domain from LexA, the ligand sensing domain from Hmox1, transactivation domain B112 and the nuclear localization signal (NLS) SV40. The linker between LexA and Hmox1 is a longer version linker (Ottoz et. al (2014) compared to sBAD, which was the reference sTF (Castaño-Cerezo et. al (2020)). FunsTF05 was codon optimised to A. niger.

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, which is interacting with LexO, that is used in FunsTF04. Hmox1 is the human heme oxygenase 1, which is the enzyme that initiates cleavage of heme (Tenhunen et. al (1969)). This enzyme was, despite the seemingly unrelated context, computationally shown to bind furfural (Santhakumar et. al (2021)).

The transactivation domain B112 is from E. coli, and 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 sTF05 and the mCherry reporter (BBa_K4129122). 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. Fluorescence from FunsTF04 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 1). FunsTF04 is funtional, but needs optimization to be compared to FunsTF05.

Figure 1: Pictures of fluorescent A. niger, which carries FunsTF04 or FunsTF05. The pictures 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
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 945
  Illegal BamHI site found at 607
  Illegal BamHI site found at 1604
  Illegal XhoI site found at 800
  Illegal XhoI site found at 1237
  Illegal XhoI site found at 1753
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]