Difference between revisions of "Part:BBa K4129105"

Revision as of 13:22, 11 October 2022

The synthetic transcription factor, FunsTF16 (LexA-SL-HbaR10-B112-SV40)

FunsTF16 is a synthetic transcription factor (sTF) based on sensor of benzoic acid derivatives (sBAD), which is a sTF in S. cerevisiae (Castaño-Cerezo et. al (2020)). FunsTF16 deviates from sBAD, because it has an nuclear localization signal (NLS) and is codon optimised to A. niger. FunsTF16 is a fusion protein consisting of the DNA-binding domain LexA, the ligand sensing domain HbaR10, transactivation domain B112 and the nuclear localization signal (NLS) SV40. In addition, was FunsTF16 condon 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)), and it is the DNA binding domain interacting with LexO that is used in FunsTF16. 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 FunsTF16 carried mutant number 10 of HbaR, which had the following mutations: A45S, L64I, A86G, A88Y, Y96A and L97G.

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)).

The designed function of FunsTF16 is to be used as a transcription factor that can initiate transcription from the 6xLexO minimal promoter (BBa_K4129115). No functionality was shown.

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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 <partinfo>BBa_K4129105 short</partinfo>
-FunsTF16 is a synthetic transcription factor (sTF) based on sensor of benzoic acid derivatives (sBAD), which is a sTF in <i>S. cerevisiae</i> (Castaño-Cerezo et. al (2020)). FunsTF16 deviates from sBAD, because it has an nuclear localization signal (NLS) and is codon optimised to <i>A. niger</i>. FunsTF16 is a fusion protein consisting of the DNA-binding domain LexA, the ligand sensing domain HbaR10, transactivation domain B112 and the nuclear localization signal (NLS) SV40.
+FunsTF16 is a synthetic transcription factor (sTF) based on sensor of benzoic acid derivatives (sBAD), which is a sTF in <i>S. cerevisiae</i> (Castaño-Cerezo et. al (2020)). FunsTF16 deviates from sBAD, because it has an nuclear localization signal (NLS) and is codon optimised to <i>A. niger</i>. FunsTF16 is a fusion protein consisting of the DNA-binding domain LexA, the ligand sensing domain HbaR10, transactivation domain B112 and the nuclear localization signal (NLS) SV40. In addition, was FunsTF16 condon optimised to <A>. <niger>.
 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)), and it is the DNA binding domain interacting with LexO that is used in FunsTF16. 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 FunsTF16 carried mutant number 10 of HbaR, which had the following mutations: A45S, L64I, A86G, A88Y, Y96A and L97G.
 The transactivation domain B112 is from <i>E. coli</i>, which were experimentally proven to initiate transcription of a synthetic promoter in <i>S. cerevisiae</i> (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)).