Difference between revisions of "Part:BBa K2012002"
 
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__NOTOC__
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<partinfo>BBa_K2012002 short</partinfo>
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<h2>BBa_K2012002 short</h2>
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Intracellular c-di-GMP concentration has been regulated by two functionally opposing enzymes, the diguanylate cyclases (DGCs) containing a GGDEF domain, and phosphodiesterases (PDEs) containing either an EAL or HD-GYP domain.
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PleD from Caulobacter crescentus, a response regulator with a diguanylate cyclase (DGC) domain.
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<img src = "https://static.igem.org/mediawiki/parts/e/e2/PleD.png" width="800"/>
 
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Mechanistic Model of PleD Regulation(Wassmann, Chan et al. 2007)<br>
 
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  The DGC domain (green) is connected via a flexible linker to the stem (receiver domain D1 [red] and adaptor domain D2 <br>[yellow]) and is supposed to be mobile relative to it. (Upper row) Activation. Phosphorylation of domain D1 leads to <br>a rearrangement of the stem domains, which, in turn, allows for formation of a tight dimeric stem (3). The dimericarrangement
 
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is a prerequisite for an efficient and productive encounter of the two substrate-loaded DGC domains to form the c-di-GMP product<br>(4). (Lower row) Product inhibition. Dimeric product molecules, (c-di-GMP)2, can crosslink the primary inhibition site on DGC, Ip,<br> with a secondary binding site either on D2, Is,D2 (5) or on the adjacent DGC domain, Is,DGC(6). The DGC domains become <br>immobilized, and the active sites are hampered from a productive encounter. Note that a possible direct communication between<br> Ip and A sites is not depicted.
 
  
  
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Reference:<hr / >
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<p>Intracellular c-di-GMP (Hengge 2009) concentration has been regulated by two functionally opposing enzymes, the diguanylate cyclases (DGCs) containing a GGDEF domain, and phosphodiesterases (PDEs) containing either an EAL or HD-GYP domain.</p>
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Wassmann, P., et al. (2007). "Structure of BeF3- -modified response regulator PleD: implications for diguanylate cyclase activation, catalysis, and feedback inhibition." Structure 15(8): 915-927.
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<p>PleD (Wassmann, Chan et al. 2007) from Caulobacter crescentus, a response regulator with a diguanylate cyclase (DGC) domain, in its activated form.</p>
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<!-- Add more about the biology of this part here-->
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<img src="https://static.igem.org/mediawiki/parts/3/3d/Congo_red_stain.png"  width="600px"/>
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===Usage and Biology===
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<p><b>Figure 1.</b> Expression of pleD. For demonstrating expression of pleD, we used Congo red staining assay. As previous mentioned, high concentration of c-di-GMP could induce E. coli synthesize exopolysaccharides, and Congo red binding is a complex phenotype that reflects various outer membrane and surface properties including the presence of adhesive structures such as curli fimbria which are involved in biofilm formation. Wild type: DE3 contain pET28b plasmid, colony which was stained red color contain pET-pleD plasmid.</p>
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<h3>Reference:</h3>
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<p>Hengge, R. (2009). "Principles of c-di-GMP signalling in bacteria." Nat Rev Microbiol 7(4): 263-273.</p>
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<p>Wassmann, P., et al. (2007). "Structure of BeF3- -modified response regulator PleD: implications for diguanylate cyclase activation, catalysis, and feedback inhibition." Structure 15(8): 915-927.</p>
  
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<span class='h3bb'>Sequence and Features</span>
 
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<h2>BBa_K2012002 SequenceAndFeatures</h2>
 
  
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<!-- Uncomment this to enable Functional Parameter display -->
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===Functional Parameters===
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<h2>BBa_K2012002 parameters</h2>
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<partinfo>BBa_K2012002 SequenceAndFeatures</partinfo>

Latest revision as of 02:52, 6 November 2016

PleD from Caulobacter crescentus, a response regulator with a diguanylate cyclase (DGC) domain.


Intracellular c-di-GMP (Hengge 2009) concentration has been regulated by two functionally opposing enzymes, the diguanylate cyclases (DGCs) containing a GGDEF domain, and phosphodiesterases (PDEs) containing either an EAL or HD-GYP domain.

PleD (Wassmann, Chan et al. 2007) from Caulobacter crescentus, a response regulator with a diguanylate cyclase (DGC) domain, in its activated form.




Figure 1. Expression of pleD. For demonstrating expression of pleD, we used Congo red staining assay. As previous mentioned, high concentration of c-di-GMP could induce E. coli synthesize exopolysaccharides, and Congo red binding is a complex phenotype that reflects various outer membrane and surface properties including the presence of adhesive structures such as curli fimbria which are involved in biofilm formation. Wild type: DE3 contain pET28b plasmid, colony which was stained red color contain pET-pleD plasmid.



Reference:

Hengge, R. (2009). "Principles of c-di-GMP signalling in bacteria." Nat Rev Microbiol 7(4): 263-273.

Wassmann, P., et al. (2007). "Structure of BeF3- -modified response regulator PleD: implications for diguanylate cyclase activation, catalysis, and feedback inhibition." Structure 15(8): 915-927.





Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 14
    Illegal BamHI site found at 1172
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 430
    Illegal NgoMIV site found at 577
    Illegal AgeI site found at 913
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 87