Difference between revisions of "Part:BBa K3071009"

 
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<partinfo>BBa_K3071009 short</partinfo>
 
<partinfo>BBa_K3071009 short</partinfo>
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===Description===
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This is the protein-encoding gene for the Phage shock protein F transcriptional activation domain fused with cAMP receptor-like protein (pspF TAD - Clp), which is a composite part constructed by ([https://parts.igem.org/Part:BBa_K3071004 BBa_K3071004]), ([https://parts.igem.org/Part:BBa_K3071008 BBa_K3071008]), amd ([https://parts.igem.org/Part:BBa_K3071007 BBa_K3071007]). This part is codon-optimized to express in express in <i>escherica coli</i> K12 strain. The western blot data of this composite part shows it could be correctly expressed in <i>E.coli</i>.
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===Biology===
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PspF ([https://parts.igem.org/Part:BBa_K3071006 BBa_K3071006])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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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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C reactive protein-like protein (Clp) ([https://parts.igem.org/Part:BBa_K3071004 BBa_K3071004]) is a global transcriptional regulator that regulates virulence factors production by activating or repressing the expression of a large set of genes in the diffusible signal factor DSF pathway. It also regulates the genes that involve in extracellular polysaccharide (EPS) synthesis, flagellum synthesis, protein, and fatty acid metabolism, multidrug resistance, iron uptake. They also regulate genes that encoding extracellular enzymes, membrane components, and a few transcription factors.
  
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C reactive protein-like protein (Clp) is a global transcriptional regulator that regulates virulence factors production by activating or repressing the expression of a large set of genes in the diffusible signal factor DSF pathway. It regulate the genes that involve in extracellular polysaccharide (EPS) synthesis, flagellum synthesis, protein and fatty acid metabolism, multidrug resistance, iron uptake. They also regulate genes that encoding extracellular enzymes, membrane components, and a few transcription factors.  
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===Usage===
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<center>[[file:T--Hong Kong-CUHK--design fig 3.png]]</center>
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<center><u>Figure 1 illustration of our synthetic biological system upon DSF activation</u></center>
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<br>
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The DNA binding domain in the C-terminus of pspF ([https://parts.igem.org/Part:BBa_K3071006 BBa_K3071006]) is replaced by the Clp to construct this fusion transcription activator for our reporter construct ([https://parts.igem.org/Part:BBa_K3071024 BBa_K3071024]). This transcription activator can activate the CBSI & II -regulated pspA promoter ([https://parts.igem.org/Part:BBa_K3071014 BBa_K3071014]) upon the diffusible signal factor (DSF) appears.  
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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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Clone 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 TAD-Clp was confirmed with successful expression at all time points. Results of blots probing Clp-pspF 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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<br>
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<center>[[T--Hong Kong-CUHK--Azathioprine treated rt-qPCR data.png]]</center>
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<center><u>Figure 3  rt-qPCR data Azathioprine treatment on the eforRED mRNA expression level </u>
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<br>
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Azathioprine is an indirect supressor to cyclic-di-GMP, treatment of azathioprine to bacteria culture can reduce the cyclic-di-GMP level and lead to activation of the pspF-Clp and by-pass the RpfC/RpfG two-component system.The result from rt-qPCR data shows that the above sysntehtic biological system is functional with significant up-regulation of reporter mRNA.
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<br>
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<center>[[file:T--Hong Kong-CUHK--DSF treatment.jpeg]]</center>
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<center><u>Figure 4  rt-qPCR data DSF treatment on the eforRED mRNA expression level</u></center>
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<br>
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The result from rt-qPCR data shows that the above sysntehtic biological system is functional with significant up-regulation of reporter mRNA upon DSF activation.  
  
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Allosterically inhibited by cyclic di-GMP (c-di-GMP), which binds to Clp and abolishes its ability to bind its target gene promoter.
 
  
  

Revision as of 05:37, 19 October 2019


Phage shock protein F transcriptional activation domain fused with Clp (pspF TAD-Clp)

Description

This is the protein-encoding gene for the Phage shock protein F transcriptional activation domain fused with cAMP receptor-like protein (pspF TAD - Clp), which is a composite part constructed by (BBa_K3071004), (BBa_K3071008), amd (BBa_K3071007). This part is codon-optimized to express in express in escherica coli K12 strain. The western blot data of this composite part shows it could be correctly expressed in E.coli.

Biology

PspF (BBa_K3071006)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.

C reactive protein-like protein (Clp) (BBa_K3071004) is a global transcriptional regulator that regulates virulence factors production by activating or repressing the expression of a large set of genes in the diffusible signal factor DSF pathway. It also regulates the genes that involve in extracellular polysaccharide (EPS) synthesis, flagellum synthesis, protein, and fatty acid metabolism, multidrug resistance, iron uptake. They also regulate genes that encoding extracellular enzymes, membrane components, and a few transcription factors.

Usage

File:T--Hong Kong-CUHK--design fig 3.png
Figure 1 illustration of our synthetic biological system upon DSF activation


The DNA binding domain in the C-terminus of pspF (BBa_K3071006) is replaced by the Clp to construct this fusion transcription activator for our reporter construct (BBa_K3071024). This transcription activator can activate the CBSI & II -regulated pspA promoter (BBa_K3071014) upon the diffusible signal factor (DSF) appears.

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)


Clone 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 TAD-Clp was confirmed with successful expression at all time points. Results of blots probing Clp-pspF 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.

T--Hong Kong-CUHK--Azathioprine treated rt-qPCR data.png
Figure 3 rt-qPCR data Azathioprine treatment on the eforRED mRNA expression level


Azathioprine is an indirect supressor to cyclic-di-GMP, treatment of azathioprine to bacteria culture can reduce the cyclic-di-GMP level and lead to activation of the pspF-Clp and by-pass the RpfC/RpfG two-component system.The result from rt-qPCR data shows that the above sysntehtic biological system is functional with significant up-regulation of reporter mRNA.

<center>File:T--Hong Kong-CUHK--DSF treatment.jpeg
Figure 4 rt-qPCR data DSF treatment on the eforRED mRNA expression level


The result from rt-qPCR data shows that the above sysntehtic biological system is functional with significant up-regulation of reporter mRNA upon DSF activation.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 907
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 910
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 907
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 907
    Illegal NgoMIV site found at 985
    Illegal AgeI site found at 90
    Illegal AgeI site found at 118
    Illegal AgeI site found at 256
  • 1000
    COMPATIBLE WITH RFC[1000]