Part:BBa_K3032117
Light-regulated plasmid copy number device (0.16) with mRFP1 (0.24)
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 medium Anderson promoter (BBa_J23110) was inserted downstream of the plasmid copy number system.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 709
Illegal NheI site found at 854
Illegal NheI site found at 877 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1463
Illegal AgeI site found at 1575 - 1000COMPATIBLE WITH RFC[1000]
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