Part:BBa_K4129108
The fungal synthetic transcription factor, FunsTF58 (LexA-LL-HbaR4-VP16-SV40)
FunsTF58 is a synthetic transcription factor (sTF). FunsTF58 should initiate the transcription through the 6xLexO minimal promoter. This sTF is the sensing part of the biosensor.
FunsTF58 is a fusion protein consisting of the DNA-binding domain LexA, the ligand sensing domain HbaR4, transactivation domain VP16 and the nuclear localization signal (NLS) SV40. The linker between LexA and HbaR4 is a longer version linker (Ottoz et. al (2014) compared to sBAD, which was the reference sTF (Castaño-Cerezo et. al (2020)). FunsTF58 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)). 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 FunsTF58 carried mutant 4 of HbaR, which had the following mutations: A45V, L69A, G71K, E77W, F85G, A86G, E87G, A88G, A89P, Y96A, L97Y, A98M, N99T, A100V, and V142Q.
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)).
FunsTF58 did not show functionality.
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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