Difference between revisions of "Part:BBa K4129107"

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]

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 <partinfo>BBa_K4129107 short</partinfo>
-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.
+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 LexA, the ligand sensing domain HbaR3, transactivation domain VP16 and the nuclear localization signal (NLS) SV40. The linker between LexA and HbaR3 is a longer version linker (Ottoz et. al (2014)  compared to sBAD, which was the reference sTF (Castaño-Cerezo et. al (2020)). FunsTF05 was codon optimised to <i>A. Niger </i>.
+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>.
 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.
 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 <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).
+<html>
+<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>
+</html>