Part:BBa_K4129103

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

FunsTF05 is a synthetic transcription factor (sTF). There are few functional synthetic transcription factors in Aspergillus niger. There is, to our knowledge, currently only one paper (Rantasalo et. al (2018)) on sTF that regulates a synthetic promoter in A. niger . The main difference between the sTF from (Rantasalo et. al (2018)) and FunsTF05 is that FunsTF05 has the possibility to become inducible.

FunsTF05 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, as seen in figure 1.

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 binding domain from Hmox1, the transactivation domain of 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 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)). 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 the domain from Hmox1, it should be able to bind to LexO site through the LexA domain and it should be able to activate transcription of a minimal promoter like 6xLexO-Pmin (BBa_K4129115) through the interaction between the transactivation domain of VP16 and the 6xLexO-Pmin. This is visualized in figure 1.

Characterization

The functionality of FunsTF05 was tested by measuring the fluorescence of A. niger expressing 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 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, carrying either BBa_K4129025 or genome integrated BBa_K3046004. 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.

The observed fluorescence of FunsTF05 was greater than that of genomically integrated BBa_K3046004 on the different plates (Figure 2). BBa_K3046004 is a strong constitutive promoter in A. niger, and this emphasises the strength of FunsTF05. The observed fluorescence of FunsTF05 was similar between plates, which indicates that FunsTF05 constitutively expresses mCherry (BBa_K4129123), thus confirming the functionality of this part.

A closely related sTF from our collection is FunsTF04 (K4129102), and the only difference is that the transactivation domain used in FunsTF04 is B112 while FunsTF05 uses that of VP16. FunsTF04 does not express mCherry to the same level as FunsTF05 and this indicates VP16 is the more active transactivation domain of B112 and VP16, when the A. niger is grown on solid plates.

Figure 3: Pictures of fluorescent A. niger, carrying 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.

In addition to characterising FunsTF05 in A. niger grown on solid media, the A. niger that expressed FunsTF05 was tested in liquid media (figure 4). 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 roughly has the highest fluorescence with FunsTF05 displaying fluorescence slightly below BBa_K3046004 at all time points (Figure 4). The fluorescence of FunsTF05 is higher than FunsTF04 and BBa_K4129025, thus further supporting that the transactivation domain of VP16 is a more active than that of B112. No induction was observed, indicated by fluorescence being either lower or within the standard deviation of the uninduced FunsTF05.

Figure 4: 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, FunsTF04 or FunsTF05. 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
  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]