Difference between revisions of "Part:BBa K3032116"

 
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<partinfo>BBa_K3032115 short</partinfo>
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The system based on re-engineered ColE1 origin of replication allowing to regulate plasmid copy number in E. coli cells with light. ColE1 plasmid replicon is based on two antisense RNA molecules: RNA I and RNA II. RNA II transcript of forms a RNA-DNA duplex with plasmid and acts as a primer for DNA polymerase. For that reason, RNA II is often called a replication initiator. However, another molecule - RNA I may bind to its antisense version of RNA II, which results in replication inhibition.
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Jayraman et al. (2016) engineered a novel bidirectional promoter system for Escherichia coli that can be induced or repressed rapidly and reversibly using the blue light dependent DNA-binding protein EL222. Inserting EL222 binding sequence between -35 and -10 promoter regions led to greater than 3-fold reduction in RFP fluorescence when the engineered E. coli was exposed to blue light.
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Vilnius-Lithuania iGEM 2019 combined RNA I/RNA II with EL222 protein to develop light-regulated system for plasmid copy number control. EL222 binding sequence was inserted between -35 and -10 regions of weak Anderson promoter (BBa_J23116), controlling transcription of the replication inhibiting – RNA I molecule. In blue light illumination EL222 protein binds to RNA I promoter. Inhibited transcription results reduced formation of RNA II-RNA I duplex and expanded plasmid copy number.
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For measurements, mRFP1 under weak Anderson promoter (0.16) was inserted downstream of the plasmid copy number system.
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3032115 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K3032115 parameters</partinfo>
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Revision as of 21:41, 16 October 2019


EL222 protein under weak Anderson (0.16) promoter (Ready for Expression)

The system based on re-engineered ColE1 origin of replication allowing to regulate plasmid copy number in E. coli cells with light. ColE1 plasmid replicon is based on two antisense RNA molecules: RNA I and RNA II. RNA II transcript of forms a RNA-DNA duplex with plasmid and acts as a primer for DNA polymerase. For that reason, RNA II is often called a replication initiator. However, another molecule - RNA I may bind to its antisense version of RNA II, which results in replication inhibition.

Jayraman et al. (2016) engineered a novel bidirectional promoter system for Escherichia coli that can be induced or repressed rapidly and reversibly using the blue light dependent DNA-binding protein EL222. Inserting EL222 binding sequence between -35 and -10 promoter regions led to greater than 3-fold reduction in RFP fluorescence when the engineered E. coli was exposed to blue light.

Vilnius-Lithuania iGEM 2019 combined RNA I/RNA II with EL222 protein to develop light-regulated system for plasmid copy number control. EL222 binding sequence was inserted between -35 and -10 regions of weak Anderson promoter (BBa_J23116), controlling transcription of the replication inhibiting – RNA I molecule. In blue light illumination EL222 protein binds to RNA I promoter. Inhibited transcription results reduced formation of RNA II-RNA I duplex and expanded plasmid copy number.

For measurements, mRFP1 under weak Anderson promoter (0.16) was inserted downstream of the plasmid copy number system.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 142
    Illegal AgeI site found at 367
  • 1000
    COMPATIBLE WITH RFC[1000]