Part:BBa_K5136042
LexRO
Biology
LexRO is a synthetic light-switchable repressor, based on a novel LOV light sensor domain, RsLOV. In the darkness, LexRO dimerizes and binds to its cognate operator sequence to repress promoter activity. Upon light exposure, the LexRO dimer dissociates, causing dissociation from the operator sequence, and initiates gene expression.
Usage and design
To prove the hrp AND gate can work. We used BBa_I0500 to construct the regulation system and obtained the composite part BBa_K4907124 and BBa_K4907125, which were assembled on the expression vector pSB1C3. And we used the pHrpL and GFP(BBa_K4907036) to construct the reporting system and obtained the composite part BBa_K4907123. Those three composite parts together form the verification system of hrp AND gate (Fig. 1) and corresponding gene circuits were transformed into E. coli DH10β for characterization.
If we use CspA Cold-responsive elements to express proteins at low temperatures, there will be a risk of gene leakage at a higher temperature than we expected because the response temperature has a broad range. To solve this problem, we plan to combine a logic AND gate with the CspA CRE. Based on it, we designed an AND gate to respond to low temperature, namely, hrp AND gate. In this system, the hrpR and hrpS genes are regulated by the CspA CRE (Fig. 2). Under low-temperature conditions, only when both proteins are expressed, can the expression of downstream genes be induced, reducing the leaky expression.
Characterization
Agarose gel electrophoresis (AGE)
When constructing this circuit of composite part BBa_K4907128 which includes the gene of HrpR, colony PCR and gene sequencing were used to verify that the transformants were correct. Target bands (1460 bp) can be observed at the position between 1000 and 1500 bp (Fig. 3).
Sequence and Features
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