Part:BBa_K4129109
The fungal synthetic transcription factor, FunsTF59 (LexA-LL-HbaR5-VP16-SV40)
FunsTF59 is a synthetic transcription factor (sTF). FunsTF59 should initiate the transcription through the 6xLexO minimal promoter. This sTF is designed to be the sensing part of the biosensor.
FunsTF59 is a fusion protein consisting of the DNA-binding domain from LexA, the ligand sensing domain from HbaR5, the transactivation domain from VP16 and the nuclear localization signal (NLS) SV40. The linker between LexA and HbaR5 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)). FunsTF59 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)). HbaR is a transcription factor from Rhodopseudomonas palustris that initiates transcription in the presence of benzoic acid (Egland. et al (2000) or in the presence of benzoic acid derivatives (Castaño-Cerezo et. al (2020)). We created 16 mutants of HbaR and FunsTF59 carried mutant 5 of HbaR, which had the following mutations: A45V, L69A, G71K, I76V, E77M, M79L, A86G, E87G, A88M, Y89A, L97P, N99T, A100V, V145Y and K148Y.
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)).
FunsTF59 did not show functionality in A. niger.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 673
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 607
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 765
- 1000COMPATIBLE WITH RFC[1000]
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