Coding

Part:BBa_K4129106

Designed by: Magnus Haahr   Group: iGEM22_DTU-Denmark   (2022-10-09)
Revision as of 11:30, 11 October 2022 by Magnus Haahr (Talk | contribs)

The fungal synthetic transcription factor, FunsTF18 (LexA-SL-HbaR12-B112-SV40)

FunsTF18 is a synthetic transcription factor (sTF). FunsTF18 should initiate the transcription through the 6xLexO minimal promoter. This sTF is the sensing part of the biosensor.

FunsTF18 is a fusion protein consisting of the DNA-binding domain: lexA, ligand sensing domain: HbaR12, transactivation domain; B112 and the nuclear localization signal (NLS) SV40. The linker between LexA and HbaR was the same as sBAD and it is annotated as short linker (SL) (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 FunsTF18. HbaR is a transcription 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 FunsTF16 carried mutant 12 of HbaR, which had the following mutations: A45S, L64I, F85M, A86G, A88Y and Y96S.

The transactivation domain B112 is from E. coli, which were 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 of the protein to the nucleus (Garcia-Bustos et. al (1991)).

Characterization

Figure 1: Pictures of fluorescent A. niger, which carries either genome integrated BBa_K3046004, BBa_K4129025 or FunsTF18. The picture are taken with 0.72 seconds exposure time. The A. niger is grown on plates containing minimal media (MM) or MM with 2 mM benzoic acid. The fluorescent is depicted as grey-white intensity.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 622
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 809
    Illegal BamHI site found at 1148
    Illegal XhoI site found at 1297
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 714
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None