Difference between revisions of "Part:BBa K4129107"
 
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=== FunsTF57 ===
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FunsTF57 is a synthetic transcription factor (sTF). FunsTF57 should initiate the transcription through the 6xLexO minimal promoter. This sTF is the sensing part of the biosensor.   
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<partinfo>BBa_K4129107 short</partinfo>
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FunsTF57 is a fusion protein consisting of the DNA-binding domain: lexA, ligand sensing domain: HbaR3, transactivation domain; VP16 and the nuclear localization signal (NLS) SV40. The linker between LexA and HbaR3 was a longer version (Ottoz et. al (2014)
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compared to sBAD (Castaño-Cerezo et. al (2020)).
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FunsTF57 is a synthetic transcription factor (sTF). FunsTF57 should initiate the transcription through the 6xLexO minimal promoter. This sTF is designed to be the sensing part of a 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 FunsTF57. 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 3 of HbaR, which had the following mutations: A45V, L69A, G71K, E77M, F85G, A86G, E87G, A88M, Y96A, L97Y, N99T, A100V.
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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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FunsTF57 is a fusion protein consisting of the DNA-binding domain from LexA, the ligand sensing domain from HbaR3, transactivation domain VP16 and the nuclear localization signal (NLS) SV40. The linker between LexA and HbaR3 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)). FunsTF57 was codon optimised to <i>A. niger </i>.
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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 FunsTF57 contained mutant 3 of HbaR, which has the following mutations: A45V, L69A, G71K, E77M, F85G, A86G, E87G, A88M, Y96A, L97Y, N99T and A100V.
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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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=== Characterization ===
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FunsTF57 had its functionality tested by growing a <i>Aspergillus niger</i>, that expresses FunsTF57, on solid media plates. The plates contained minimal media, minimal media with 2 mM benzoic acid or minimal media with 0.6 g/L furfural. The fluorescence of the plates was assessed, after four days of incubation at 30<span>&#8451;</span>, using the Vilber Fusion FX imager system. The intensity of the fluorescences was presented as grey-white. The <i>A. niger</i> FunsTF57 displayed no fluorescence on any of the plates (figure 1).
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<figure><img style="width: 60%; padding:28px;"src="https://static.igem.org/mediawiki/parts/1/1b/SES_FunsTF02_and_57.png" class="safetyfirstimg"><figcaption>Figure 1: Pictures of fluorescent <i>A</i>. <i>niger</i>, carrying either FunsTF02 or FunsTF57. The picture are taken with 1.04 seconds exposure time. The <i>A</i>. <i>niger</i> is grown on plates containing minimal media (MM), MM with 2 mM benzoic acid or MM with 0.6 g/L furfural. The fluorescent is depicted as grey-white intensity.</figcaption></figure>
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Latest revision as of 20:36, 11 October 2022

The fungal synthetic transcription factor, FunsTF57 (LexA-LL-HbaR3-VP16-SV40)

FunsTF57 is a synthetic transcription factor (sTF). FunsTF57 should initiate the transcription through the 6xLexO minimal promoter. This sTF is designed to be the sensing part of a biosensor.

FunsTF57 is a fusion protein consisting of the DNA-binding domain from LexA, the ligand sensing domain from HbaR3, transactivation domain VP16 and the nuclear localization signal (NLS) SV40. The linker between LexA and HbaR3 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)). FunsTF57 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 FunsTF57 contained mutant 3 of HbaR, which has the following mutations: A45V, L69A, G71K, E77M, F85G, A86G, E87G, A88M, Y96A, L97Y, N99T and A100V.

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)).

Characterization

FunsTF57 had its functionality tested by growing a Aspergillus niger, that expresses FunsTF57, on solid media plates. The plates contained minimal media, minimal media with 2 mM benzoic acid or minimal media with 0.6 g/L furfural. The fluorescence of the plates was assessed, after four days of incubation at 30℃, using the Vilber Fusion FX imager system. The intensity of the fluorescences was presented as grey-white. The A. niger FunsTF57 displayed no fluorescence on any of the plates (figure 1).

Figure 1: Pictures of fluorescent A. niger, carrying either FunsTF02 or FunsTF57. The picture are taken with 1.04 seconds exposure time. The A. niger is grown on plates containing minimal media (MM), MM with 2 mM benzoic acid or MM with 0.6 g/L furfural. The fluorescent is depicted as grey-white intensity.


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]