Difference between revisions of "Part:BBa K3071007"

 
 
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<partinfo>BBa_K3071007 short</partinfo>
 
<partinfo>BBa_K3071007 short</partinfo>
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===Descirption===
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This the protein-encoding gene for the Transcription activation domain of Phage-shock protein pspF (PspF TAD), which is originated from <i>Escherica coli</i> K12 strain.  The SDS-PAGE gel data and western blot data of our composite part (<html><a href="https://parts.igem.org/Part:BBa_K3071020">BBa_K3071020</a></html>) show it could be correctly expressed in <i>Escherica coli</i> Bl21(DE3) strain in form of the fusion protein. In our project, the original DNA-binding domain would be replaced by the Clp, leaving the transcription activation domain(TAD) in our biobrick design (<html><a href="https://parts.igem.org/Part:BBa_K3071009">BBa_K3071009</a></html>).
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===Biology===
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<center>[[file:T--Hong_Kong-CUHK--pspF_structure.png]]</center>
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<center><u>Figure 1 Protein structure of PspF</u></center>
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<br>
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PspF has a hexameric structure, with α/β and α domains in each monomer (figure 1). It has ATPase activity in E. Coli to promote DNA strand separation, forming the open complex. Loop 1 (L1) and loop 2 (L2) are two loops locate in the α/β domains and α domains respectively. They are responsible for the interaction between PspF and the sigma 54. The 8 to 238 amino acids are the Sigma 54 interactive domain while the DNA strand binding motif is at the amino acid position 302 to 321.
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===Usage===
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PspF is a constitutively active enhancer-binding protein for activating the PspA promoter (<html><a href="https://parts.igem.org/Part:BBa_K3071013">BBa_K3071013</a></html>) transcription in vivo. The enhancer is located in -80 to -126 upstream of the pspA transcription start site.
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===Characterization===
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<center>[[file:T--Hong_Kong-CUHK--Clp-pspF_12-28hr_westernblot.png]]</center>
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<center><u>Figure 2  Western blot analysis of pspF-Clp protein expression using Myc-Tag (9B11) Mouse mAb (1:2000)</u></center>
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<br>
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The bacteria culture was induced by 1mM IPTG at 32℃  and collected at different time points, which are 12hr, 16hr, 20hr, 24hr, and 28hr. After blotting with corresponding antibodies, pspF-Clp was confirmed with successful expression at all time points. Results of blots probing pspF-Clp showed that clones collected at 12hr contained the highest amount of target proteins and the protein quantity decreased from 16hr to 28hr. This may due to degradation inside the cells.
  
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PspF is a constitutively active enhancer-binding protein for activating the PspA operon transcription in vivo. The enhancer is located in -80 to -126 upstream of the pspA transcription start site. It has a hexameric structure, with &#945;/&#946; and &#945; domains in each monomer. It has ATPase activity in E. Coli to promote DNA strand separation, forming the open complex. Loop 1 (L1) and loop 2 (L2) are two loops locate in the &#945;/&#946; domains and &#945; domains respectively. They are responsible for the interaction between PspF and the sigma 54. The 8 to 238 amino acids are the Sigma 54 interactive domain while the DNA strand binding motif is at the amino acid position 302 to 321. In our project, the original DNA-binding domain would be replaced by the Clp, leaving the transcription activation domain(TAD) in our biobrick design.
 
  
 
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Latest revision as of 08:16, 21 October 2019


Phage shock protein F transcriptional activation domain (PspF TAD)

Descirption

This the protein-encoding gene for the Transcription activation domain of Phage-shock protein pspF (PspF TAD), which is originated from Escherica coli K12 strain. The SDS-PAGE gel data and western blot data of our composite part (BBa_K3071020) show it could be correctly expressed in Escherica coli Bl21(DE3) strain in form of the fusion protein. In our project, the original DNA-binding domain would be replaced by the Clp, leaving the transcription activation domain(TAD) in our biobrick design (BBa_K3071009).

Biology

T--Hong Kong-CUHK--pspF structure.png
Figure 1 Protein structure of PspF


PspF has a hexameric structure, with α/β and α domains in each monomer (figure 1). It has ATPase activity in E. Coli to promote DNA strand separation, forming the open complex. Loop 1 (L1) and loop 2 (L2) are two loops locate in the α/β domains and α domains respectively. They are responsible for the interaction between PspF and the sigma 54. The 8 to 238 amino acids are the Sigma 54 interactive domain while the DNA strand binding motif is at the amino acid position 302 to 321.

Usage

PspF is a constitutively active enhancer-binding protein for activating the PspA promoter (BBa_K3071013) transcription in vivo. The enhancer is located in -80 to -126 upstream of the pspA transcription start site.

Characterization

T--Hong Kong-CUHK--Clp-pspF 12-28hr westernblot.png
Figure 2 Western blot analysis of pspF-Clp protein expression using Myc-Tag (9B11) Mouse mAb (1:2000)


The bacteria culture was induced by 1mM IPTG at 32℃ and collected at different time points, which are 12hr, 16hr, 20hr, 24hr, and 28hr. After blotting with corresponding antibodies, pspF-Clp was confirmed with successful expression at all time points. Results of blots probing pspF-Clp showed that clones collected at 12hr contained the highest amount of target proteins and the protein quantity decreased from 16hr to 28hr. This may due to degradation inside the cells.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 90
    Illegal AgeI site found at 118
    Illegal AgeI site found at 256
  • 1000
    COMPATIBLE WITH RFC[1000]