Difference between revisions of "Part:BBa K4129111"

Revision as of 21:14, 11 October 2022

The fungal synthetic transcription factor, FunsTF62 (LexA-LL-HbaR8-VP16-SV40)

FunsTF62 is a synthetic transcription factor (sTF). FunsTF62 should initiate the transcription through the 6xLexO minimal promoter. This sTF is designed to be the sensing part of the biosensor.

FunsTF62 is a fusion protein consisting of the DNA-binding domain from LexA, the ligand sensing domain from HbaR8, the transactivation domain from VP16 and the nuclear localization signal (NLS) SV40. The linker between LexA and HbaR8 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)). FunsTF62 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 FunsTF62 carried mutant 8 of HbaR, which had the following mutations: A45V, L69A, G71K, E77A, A86G, E89G, A90G, A91P, Y96A, L97Y, A98S, N99T, A100V and V145Y.

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)).

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 673
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 607
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 765
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K4129111 short</partinfo>
-FunsTF62 is a synthetic transcription factor (sTF). FunsTF62 should initiate the transcription through the 6xLexO minimal promoter. This sTF is the sensing part of the biosensor.
+FunsTF62 is a synthetic transcription factor (sTF). FunsTF62 should initiate the transcription through the 6xLexO minimal promoter. This sTF is designed to be the sensing part of the biosensor.
-FunsTF62 is a fusion protein consisting of the DNA-binding domain LexA, the ligand sensing domain HbaR8, transactivation domain VP16 and the nuclear localization signal (NLS) SV40. The linker between LexA and HbaR8 is a longer version linker (Ottoz et. al (2014)  compared to sBAD, which was the reference sTF (Castaño-Cerezo et. al (2020)). FunsTF62 was codon optimised to <i>A. Niger </i>.
+FunsTF62 is a fusion protein consisting of the DNA-binding domain from LexA, the ligand sensing domain from HbaR8, the transactivation domain from VP16 and the nuclear localization signal (NLS) SV40. The linker between LexA and HbaR8 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)). FunsTF62 was codon optimised to <i>A. niger</i>.
 LexA is a repressor that regulates the SOS response in <i>E. coli</i> (Radman. 1975). LexA binds to a specific DNA motif, namely LexO sites (Erill. et al (2003)). HbaR is a transcription factor from <i>Rhodopseudomonas palustris</i> 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 FunsTF62 carried mutant 8 of HbaR, which had the following mutations: