Difference between revisions of "Part:BBa K1689015"

 
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<partinfo>BBa_K1689015 short</partinfo>
 
<partinfo>BBa_K1689015 short</partinfo>
  
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pSB1C3-6His-F[1,2]-dCas9, segment F[1,2] fusion with dCas9, 6His tag allows for protein purification.
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F[1,2] segment of DHFR fused with dCas9<br/>
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Dihydrofolate reductase(DHFR) exists in all kinds of organisms, which consists the adenine-binding domain (fragment [2]) and a discontinuous domain (fragment [I]/[3]). Fragments [1] and [2] contain folate-binding pocket and the NADPH-binding groove while fragment [3] has few crystal contacts with the substrates.
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DHFR catalyzes the NADPH-dependent reduction of dihydrofolate (H2folate) or folic acid to tetrahydrofolate (H4folate), tetrahydrofolate and its derivatives are essential for purin and thymidylate synthesis, necessary for cell division and growth.  DHFR can be used for proetin fragment complementary assay(PCA), only reconstituted DHFR in the presence of inducer allows cell growth. The activity of DHFR can also be determined spectrophotometrically ,  with absorbance change at 340nm.<br/>
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Here we choose to cleave EcDHFR at position 88 so as not to disrupt the active site and NADPH cofactor-binding sites, with the  reduction of dihydrofolate (H2folate) or folic acid to tetrahydrofolate (H4folate), we could detect electrochemical signals immediately.<br/>
  
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https://static.igem.org/mediawiki/2015/0/0a/Peking-Hardware_From_Bioluminescence_to_Electronic_signal.gif<br/><br/><br/>
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Revision as of 18:29, 18 September 2015

F[1,2]-dCas9 

F[1,2] segment of DHFR fused with dCas9

Dihydrofolate reductase(DHFR) exists in all kinds of organisms, which consists the adenine-binding domain (fragment [2]) and a discontinuous domain (fragment [I]/[3]). Fragments [1] and [2] contain folate-binding pocket and the NADPH-binding groove while fragment [3] has few crystal contacts with the substrates. DHFR catalyzes the NADPH-dependent reduction of dihydrofolate (H2folate) or folic acid to tetrahydrofolate (H4folate), tetrahydrofolate and its derivatives are essential for purin and thymidylate synthesis, necessary for cell division and growth. DHFR can be used for proetin fragment complementary assay(PCA), only reconstituted DHFR in the presence of inducer allows cell growth. The activity of DHFR can also be determined spectrophotometrically , with absorbance change at 340nm.

Here we choose to cleave EcDHFR at position 88 so as not to disrupt the active site and NADPH cofactor-binding sites, with the reduction of dihydrofolate (H2folate) or folic acid to tetrahydrofolate (H4folate), we could detect electrochemical signals immediately.

Peking-Hardware_From_Bioluminescence_to_Electronic_signal.gif


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1441
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 4
    Illegal BamHI site found at 3720
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]