Difference between revisions of "Part:BBa K4129101"
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=== Characterization ===
 
=== Characterization ===
  
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The functionally of FunsTF05 was tested by measuring the fluorescence of an <i>A. niger</i> carring FunsTF05 and the mCherry reporter (BBa_K4129123). 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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<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: This is the figure text.</figcaption></figure>
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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 genome 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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Revision as of 08:58, 11 October 2022

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

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 deviates from sBAD, because it has an nuclear localization signal (NLS) and is codon optimised to A. niger. FunsTF02 is a fusion protein consisting of the DNA-binding domain LexA, the ligand sensing domain 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 can 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)), and it is the DNA binding domain interacting with LexO that is used in FunsTF02. 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, which 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 FunsTF05 was tested by measuring the fluorescence of an A. niger carring FunsTF05 and the mCherry reporter (BBa_K4129123). 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 genome 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.



Figure 2: This is the figure text.


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]