Difference between revisions of "Part:BBa K4129114"

 
Line 1: Line 1:
 +
__NOTOC__
 +
<partinfo>BBa_K4129114 short</partinfo>
  
 +
FunsTF70 is a synthetic transcription factor (sTF).  FunsTF05 should function as a transcription factor that can initiate transcription of the 6xLexO minimal promoter (BBa_K4129115). This sTF will be the sensing part of the biosensor.
 +
 +
FunsTF70 is a fusion protein consisting of the DNA-binding domain: lexA, ligand sensing domain: HbaR16, transactivation domain; VP16 and the nuclear localization signal (NLS) SV40. The linker between LexA and HbaR16 was a longer version (Ottoz et. al (2014) compared to sBAD (Castaño-Cerezo et. al (2020)).
 +
 +
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 FunsTF70. 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)). We created 16 mutants of HbaR and FunsTF70 carried mutant 16 of HbaR, which had the following mutations: L64I, F85H, A86G, A90Y and L97G.
 +
 +
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)).
 +
 +
=== Characterization ===
 +
 +
The functionally of sTF05 were tested by measuring the fluorescence of an <i>A. niger</i> carring sTF05 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.
 +
 +
The fluorescence was assessed using the Vilber Fusion FX imager system to measure fluorescence shown as grey-white intensity. The exposure time was normalised to the fluorescences from genomic integrated BBa_K3046004, and this enabled comparison between plates. The exposure time used was 0.72 seconds.
 +
It is observed that genomic integrated BBa_K3046004 displays fluorescence and the negative control of BBa_K4129025 did not. It should be noted that no growth was observed on the plates containing 0.6 g/L furfural (figure 1).
 +
 +
 +
<html>
 +
<figure><img style="width: 60%; padding:28px;"src="https://static.igem.org/mediawiki/parts/b/ba/FunsTF70_and_control_on_plates.png" class="safetyfirstimg"><figcaption>Figure 1: Pictures of fluorescent <i>A</i>. <i>niger</i>, which carries either genome integrated BBa_K3046004, BBa_K4129025 or FunsTF70. The picture are taken with 0.72 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>
 +
 +
 +
 +
<!-- -->
 +
<span class='h3bb'>Sequence and Features</span>
 +
<partinfo>BBa_K4129103 SequenceAndFeatures</partinfo>
 +
 +
 +
<!-- Uncomment this to enable Functional Parameter display
 +
===Functional Parameters===
 +
<partinfo>BBa_K4129103 parameters</partinfo>
 +
<!-- -->

Revision as of 20:12, 10 October 2022

The fungal synthetic transcription factor, FunsTF70 (LexA-LL-HbaR16-VP16-SV40)

FunsTF70 is a synthetic transcription factor (sTF). FunsTF05 should function as a transcription factor that can initiate transcription of the 6xLexO minimal promoter (BBa_K4129115). This sTF will be the sensing part of the biosensor.

FunsTF70 is a fusion protein consisting of the DNA-binding domain: lexA, ligand sensing domain: HbaR16, transactivation domain; VP16 and the nuclear localization signal (NLS) SV40. The linker between LexA and HbaR16 was a longer version (Ottoz et. al (2014) compared to sBAD (Castaño-Cerezo et. al (2020)).

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 FunsTF70. 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 FunsTF70 carried mutant 16 of HbaR, which had the following mutations: L64I, F85H, A86G, A90Y and L97G.

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)).

Characterization

The functionally of sTF05 were tested by measuring the fluorescence of an A. niger carring sTF05 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 was assessed using the Vilber Fusion FX imager system to measure fluorescence shown as grey-white intensity. The exposure time was normalised to the fluorescences from genomic integrated BBa_K3046004, and this enabled comparison between plates. The exposure time used was 0.72 seconds. It is observed that genomic integrated BBa_K3046004 displays fluorescence and the negative control of BBa_K4129025 did not. It should be noted that no growth was observed on the plates containing 0.6 g/L furfural (figure 1).


Figure 1: Pictures of fluorescent A. niger, which carries either genome integrated BBa_K3046004, BBa_K4129025 or FunsTF70. The picture are taken with 0.72 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
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 945
    Illegal BamHI site found at 607
    Illegal XhoI site found at 800
    Illegal XhoI site found at 1237
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]