Difference between revisions of "Part:BBa K4129109"
 
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=== FunsTF59 ===
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__NOTOC__
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FunsTF59 is a synthetic transcription factor (sTF). FunsTF59 should initiate the transcription through the 6xLexO minimal promoter. This sTF is the sensing part of the biosensor.   
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<partinfo>BBa_K4129109 short</partinfo>
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FunsTF59 is a fusion protein consisting of the DNA-binding domain: lexA, ligand sensing domain: HbaR5, transactivation domain; VP16 and the nuclear localization signal (NLS) SV40. The linker between LexA and HbaR5 was a longer version (Ottoz et. al (2014)
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compared to sBAD (Castaño-Cerezo et. al (2020)).
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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.  
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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 FunsTF59. HbaR is a transcriptional 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 FunsTF57 carried mutant 5 of HbaR, which had the following mutations:  
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A45V, L69A, G71K, I76V, E77M, M79L, A86G, E87G, A88M, Y89A,L97P,N99T,A100V, V145Y and K148Y.
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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 <i>Aspergillus niger</i>.
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Viral Protein 16 (VP16) from herpes simplex virus type 1 is a transcription factor that uses a transactivation domain to recruit the RNA polymerase II.The NLS SV40 is a small peptide sequence of PKKKRKV that enables transport of the protein to the nucleus (Garcia-Bustos et. al (1991)).
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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 FunsTF59 carried mutant 5 of HbaR, which had the following mutations:  
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A45V, L69A, G71K, I76V, E77M, M79L, A86G, E87G, A88M, Y89A, L97P, N99T, A100V, V145Y and K148Y.
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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)).
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FunsTF59 did not show functionality in <i>A. niger</i>.
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4129103 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K4129109 SequenceAndFeatures</partinfo>
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K4129103 parameters</partinfo>
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<partinfo>BBa_K4129109 parameters</partinfo>
 
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Latest revision as of 20:47, 11 October 2022

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


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]