Difference between revisions of "Part:BBa K4129101"
 
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=== FunsTF02 ===
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FunsTF02 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)). FunsTF02 should initiate the transcription through the 6xLexO minimal promoter. This sTF is the sensing part of the biosensor.
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<partinfo>BBa_K4129101 short</partinfo>
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FunsTF02 is a fusion protein consisting of the DNA-binding domain: lexA, ligand sensing domain: HbaR, transactivation domain; B112 and the nuclear localization signal (NLS) SV40. The linker between LexA and HbaR was the same as sBAD (SL) (Castaño-Cerezo et. al (2020)).
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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, lexO (Erill. et al (2003)), and it is the DNA binding domain that interacts with LexO that is used in FunsTF02. HbaR is a transcriptional factor from <i>Rhodopseudomonas palustris</i> 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)).The transactivation domain B112 is from <i>E. coli</i>, 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 og the protein to the nucleus (Garcia-Bustos et. al (1991)).
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FunsTF02 is a synthetic transcription factor (sTF) based on a sensor of benzoic acid derivatives (sBAD), which is a sTF in <i>S. cerevisiae</i> (Castaño-Cerezo et. al (2020)). FunsTF02 deviates from sBAD, in that it has a nuclear localization signal (NLS) and is codon optimised to <i>A. niger</i>. FunsTF02 is a fusion protein consisting of the DNA-binding domain from LexA, the ligand sensing domain from HbaR, transactivation domain B112 and the nuclear localization signal (NLS) SV40.
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The designed function of FunsTF02 is to be used as a transcription factor that can initiate transcription from the 6xLexO minimal promoter (BBa_K4129115). 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 <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)).
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The transactivation domain B112 is from <i>E. coli</i>, and it was 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 og the protein to the nucleus (Garcia-Bustos et. al (1991)).
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=== Characterization ===
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The functionally of FunsTF02 was tested by measuring the fluorescence of an <i>A. niger</i> expressing FunsTF02 and the mCherry reporter (BBa_K4129121). The <i>A. niger</i> is grown on solid media plates. The plates contained either minimal media, minimal media with mM benzoic acid or MM with 0.6 g/L furfural.
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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 exposure time was normalised to the fluorescences from genomically integrated BBa_K3046004. It is observed that genomically integrated BBa_K3046004 displays fluorescence and the negative control of BBa_K4129025 did not (figure 1).
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<html>
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<figure><img style="width: 60%; padding:28px;"src="https://static.igem.org/mediawiki/parts/1/17/SES_Control_ET104.png" class="safetyfirstimg"><figcaption>Figure 1: Pictures of fluorescent <i>A</i>. <i>niger</i>, which carries either BBa_K4129025 or genomically integrated BBa_K3046004. 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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</html>
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Fluorescence from FunsTF02 is only obsvered when the <i>A. niger</i> was grown minimal media. In addition was the fluorescence is localised to the perimeter of the colony and displayed as low intensity (figure 2). FunsTF02 is funtional.
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<html>
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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 2: Pictures of fluorescent <i>A</i>. <i>niger</i>, which carries 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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</html>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4129101 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K4129101 parameters</partinfo>
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Latest revision as of 12:43, 12 October 2022

The fungal synthetic transcription factor, FunsTF02 (LexA-SL-HbaR-B112-SV40)

FunsTF02 is a synthetic transcription factor (sTF) based on a sensor of benzoic acid derivatives (sBAD), which is a sTF in S. cerevisiae (Castaño-Cerezo et. al (2020)). FunsTF02 deviates from sBAD, in that it has a nuclear localization signal (NLS) and is codon optimised to A. niger. FunsTF02 is a fusion protein consisting of the DNA-binding domain from LexA, the ligand sensing domain from HbaR, transactivation domain B112 and the nuclear localization signal (NLS) SV40.

The designed function of FunsTF02 is to be used as a transcription factor that can initiate transcription from the 6xLexO minimal promoter (BBa_K4129115). This sTF is designed to be the sensing part of a biosensor.

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)).

The transactivation domain B112 is from E. coli, and it was 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 og the protein to the nucleus (Garcia-Bustos et. al (1991)).


Characterization

The functionally of FunsTF02 was tested by measuring the fluorescence of an A. niger expressing FunsTF02 and the mCherry reporter (BBa_K4129121). The A. niger is grown on solid media plates. The plates contained either minimal media, minimal media with mM benzoic acid or MM 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 exposure time was normalised to the fluorescences from genomically integrated BBa_K3046004. It is observed that genomically integrated BBa_K3046004 displays fluorescence and the negative control of BBa_K4129025 did not (figure 1).


Figure 1: Pictures of fluorescent A. niger, which carries either BBa_K4129025 or genomically integrated BBa_K3046004. 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.

Fluorescence from FunsTF02 is only obsvered when the A. niger was grown minimal media. In addition was the fluorescence is localised to the perimeter of the colony and displayed as low intensity (figure 2). FunsTF02 is funtional.

Figure 2: Pictures of fluorescent A. niger, which carries 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 622
  • 21
    INCOMPATIBLE WITH RFC[21]
    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]