Difference between revisions of "Part:BBa K4129103"
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<partinfo>BBa_K4129103 short</partinfo>
 
<partinfo>BBa_K4129103 short</partinfo>
  
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FunsTF05 is a synthetic transcription factor (sTF). FunsTF05 should initiate the transcription through the 6xLexO minimal promoter. This sTF is the sensing part of the biosensor.  
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FunsTF05 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.
 
   
 
   
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FunsTF05 is a fusion protein consisting of the DNA-binding domain: lexA, ligand sensing domain: Hmox1, transactivation domain; VP16 and the nuclear localization signal (NLS) SV40. The linker between LexA and Hmox1 was a longer version (Ottoz et. al (2014)
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FunsTF05 is a fusion protein consisting of the DNA-binding domain: LexA, ligand sensing domain: Hmox1, transactivation domain; VP16 and the nuclear localization signal (NLS) SV40. The linker between LexA and Hmox1 was a longer version (Ottoz et. al (2014)
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compared to sBAD (Castaño-Cerezo et. al (2020)).
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compared to sBAD, which was the reference sTF (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 FunsTF05. Hmox1 is the human heme oxygenase 1, which is the enzyme that initiates cleavage of heme (Tenhunen et al. (1969)). This function is not of our interest, but it was computationally shown that furfural binds to hmox1 (Santhakumar et al (2021)).
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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 (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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<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>Aspergillus</i>. <i>niger</i>, which carries either BBa_K4129025 or genome integrated BBa_K3046004. The <i>Aspergillus</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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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 FunsTF05. Hmox1 is the human heme oxygenase 1, which can cleave heme. This function is not of our interest, but it was shown that furfural binds to hmox1.
 
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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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<figure><img style="width: 60%; padding:28px;"src="https://static.igem.org/mediawiki/parts/c/c1/FunsTF05_all_plates.png" class="safetyfirstimg"><figcaption>Figure 1: <i>Aspergillus</i>. <i>niger</i> grown on minimal media (MM), MM with 2 mM benzoic acid and MM with 0.6 g/L furfural .</figcaption></figure>
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<figure><img style="width: 60%; padding:28px;"src="https://static.igem.org/mediawiki/parts/c/c1/FunsTF05_all_plates.png" class="safetyfirstimg"><figcaption>Figure 2: Pictures of fluorescent <i>Aspergillus</i>. <i>niger</i>, which carries FunsTF05. The <i>Aspergillus</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 19:26, 10 October 2022

The synthetic transcription factor, FunsTF05 (LexA-LL-Hmox1-VP16-SV40)

FunsTF05 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.

FunsTF05 is a fusion protein consisting of the DNA-binding domain: LexA, ligand sensing domain: Hmox1, transactivation domain; VP16 and the nuclear localization signal (NLS) SV40. The linker between LexA and Hmox1 was a longer version (Ottoz et. al (2014) compared to sBAD, which was the reference sTF (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 FunsTF05. Hmox1 is the human heme oxygenase 1, which is the enzyme that initiates cleavage of heme (Tenhunen et al. (1969)). This function is not of our interest, but it was computationally shown that furfural binds to hmox1 (Santhakumar et al (2021)).

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 (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

Figure 1: Pictures of fluorescent Aspergillus. niger, which carries either BBa_K4129025 or genome integrated BBa_K3046004. The Aspergillus. 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: Pictures of fluorescent Aspergillus. niger, which carries FunsTF05. The Aspergillus. 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]