Difference between revisions of "Part:BBa K346000"

 
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<partinfo>BBa_K346000 short</partinfo>
 
<partinfo>BBa_K346000 short</partinfo>
  
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This part is the coding region of MerR, which is a mercury responsive regulator from Tn21.
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T3 polymerase can activate the expression of different T3 promoters in T3 phage. Since during different time the phage need different expression level of different genes, there must be T3 promoters of variable sensitivity.
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In our experiment, we constructed 14 different T3 promoter by primer annealing and by standard digestion to put it into standard plasmid. We then add GFP downstream to function as a reporter. By transforming another plasmid contains T7 promoter+RBS+T3 polymerase, we were able to detect the expression level of different promoters. We finally constructed 9 promoters with variable sensitivity, and put them in order according to the GFP expression level. We found that among them the Phi9 T3 promoter has a medium expression level and was more robust in the system, so we chose this one to be used in our kit.
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<!-- Add more about the biology of this part here
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===Usage and Biology===
 
===Usage and Biology===
  

Revision as of 01:31, 23 October 2010

RBS(B0032)+T3 DNA-directed RNA polymerase

T3 polymerase can activate the expression of different T3 promoters in T3 phage. Since during different time the phage need different expression level of different genes, there must be T3 promoters of variable sensitivity. In our experiment, we constructed 14 different T3 promoter by primer annealing and by standard digestion to put it into standard plasmid. We then add GFP downstream to function as a reporter. By transforming another plasmid contains T7 promoter+RBS+T3 polymerase, we were able to detect the expression level of different promoters. We finally constructed 9 promoters with variable sensitivity, and put them in order according to the GFP expression level. We found that among them the Phi9 T3 promoter has a medium expression level and was more robust in the system, so we chose this one to be used in our kit. 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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2585