Part:BBa_K4129103

The synthetic transcription factor, FunsTF05 (LexA-LL-Hmox1-VP16-SV40)

FunsTF05 is a synthetic transcription factor (sTF). FunsTF05 is designed to function as a transcription factor. In theory, FunsTF05 should be able to interact with an inducer and this interaction will facilitate transcription from a promoter. This design of sTF is a possible sensing part of a biosensor.

Figure 1: The proposed mode of action of the synthetic expression system with FunsTF05. Funs05 consisting of LexA (orange), Hmox1 (green) and VP16 (pink) interacts with a inducer. This interacting promotes expression of mCherry 6xLexO-Pmin.

FunsTF05 is a fusion protein consisting of the DNA-binding domain from LexA, the ligand sensing domain from Hmox1, transactivation domain VP16 and the nuclear localization signal (NLS) SV40. The linker between the LexA domain and the Hmox1 domain is a longer linker (Ottoz et. al (2014)) compared to the linker used in sBAD, which was the reference sTF (Castaño-Cerezo et. al (2020)). FunsTF05 was codon optimised to Aspergillus. 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)). 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)).

Viral Protein 16 (VP16) from Herpes simplex virus type 1 is a transcription factor with a transactivation domain that recruits RNA polymerase II (Hirai et al. (2010)).The NLS SV40 is a small peptide sequence of PKKKRKV that enables transport of the protein to the nucleus (Garcia-Bustos et. al (1991)).

In short, FunsTF05 should be able to interact with furfural due to Hmox1, it should be able to bind to LexO site, and it should be able to activate transcription of a minimal promoter like 6xLexO-Pmin (BBa_K4129103).

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 2).

Figure 2: 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.

The observed fluorescence of FunsTF05 was greater than that of genomically integrated BBa_K3046004 on the different plates. BBa_K3046004 is a strong constitutive promoter in A. niger, and this emphasises the strength of FunsTF05. The observed fluorescence of FunsTF05 did not differ too much between plates, which indicates that FunsTF05 constitutively expresses mCherry (BBa_K4129123) and thus confirming the functionality of this part (figure 3).

A closely related sTF from our collection is FunsTF04 (K4129102), and the only difference is that the transactivation domain used in FunsTF04 is B112 and FunsTF05 uses VP16. FunsTF04 does not express mCherry to the same level as FunsTF05 and this indicates VP16 is the better transactivation domain than B112.

Figure 3: 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 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]