Difference between revisions of "Part:BBa K1886016"
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<partinfo>BBa_K1886016 parameters</partinfo>
 
<partinfo>BBa_K1886016 parameters</partinfo>
 
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<h2>'''Characterization'''</h2>
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<h3> BACKGROUND </h3>
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<h4>Overview</h4>
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We simplified the three-plasmid system based on our reference by constructing all the segments in one plasmid. And we tested in E.coli MG1655. We used arabinose promoter (PBAD) and lactose promoter (Plac) as inducers and we have verified the corresponding concentration and time.
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<br>
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<h4>Principle</h4>
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This is the input part of AND GATE. After being induced by IPTG, plac promoter activates the expression of T7 RNA polymerase which contains two amber mutations (T7 ptag) . After being induced by L-arabinose, pbad promoter activates the expression of a special tRNA (supD), which is used to identify amber mutation. In the wild-type E.coli, amber mutation will inhibit the translation of T7 RNA polymerase. Therefore, only when the two inputs exist together, T7 RNA polymerase can be expressed, and then T7 promoter can be activated to express the downstream gene.
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<br>
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<h3>RESULTS</h3>
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<br>
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<h4>Gel electrophoretic analysis</h4>
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<br>
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[[File:RIRaiiA1016.jpg|800px|thumb|left|Fig.1 Fig.1 Gel electrophoretic analyses of PCR products ]]
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<br>
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<br>
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<h4>stable oscillation in microfluidics chips</h4>
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[[File:Oscillationa.png|800px|thumb|left|Fig.2 Fig.2  ]]
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<br>
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<br>
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<br>

Revision as of 00:40, 20 October 2016


AND GATE-input_ara+IPTG

This is the input part of AND GATE. After being induced by IPTG, plac promoter activates the expression of T7 RNA polymerase which contains two amber mutations (T7 ptag) . After being induced by L-arabinose, pbad promoter activates the expression of a special tRNA (supD), which is used to identify amber mutation. In the wild-type E.coli, amber mutation will inhibit the translation of T7 RNA polymerase. Therefore, only when the two inputs exist together, T7 RNA polymerase can be expressed, and then T7 promoter can be activated to express the downstream gene.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 171
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 174
    Illegal BsaI.rc site found at 4155


Characterization

BACKGROUND

Overview

We simplified the three-plasmid system based on our reference by constructing all the segments in one plasmid. And we tested in E.coli MG1655. We used arabinose promoter (PBAD) and lactose promoter (Plac) as inducers and we have verified the corresponding concentration and time.

Principle

This is the input part of AND GATE. After being induced by IPTG, plac promoter activates the expression of T7 RNA polymerase which contains two amber mutations (T7 ptag) . After being induced by L-arabinose, pbad promoter activates the expression of a special tRNA (supD), which is used to identify amber mutation. In the wild-type E.coli, amber mutation will inhibit the translation of T7 RNA polymerase. Therefore, only when the two inputs exist together, T7 RNA polymerase can be expressed, and then T7 promoter can be activated to express the downstream gene.

RESULTS


Gel electrophoretic analysis


File:RIRaiiA1016.jpg

stable oscillation in microfluidics chips

Fig.2 Fig.2