Difference between revisions of "Part:BBa K5136042"
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===Usage and design===
 
===Usage and design===
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To prove the <i>hrp</i> AND gate can work. We used <partinfo>BBa_I0500</partinfo> to construct the regulation system and obtained the composite part <partinfo>BBa_K4907124</partinfo> and <partinfo>BBa_K4907125</partinfo>, which were assembled on the expression vector pSB1C3. And we used the pHrpL and GFP(<partinfo>BBa_K4907036</partinfo>) to construct the reporting system and obtained the composite part <partinfo>BBa_K4907123</partinfo>. Those three composite parts together form the verification system of <i>hrp</i> AND gate (Fig. 1) and corresponding gene circuits were transformed into <i>E. coli</i> DH10β for characterization.
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In the darkness, LexRO dimerizes and binds to the cognate operator sequence to repress the activity of pColE408. Upon blue light exposure, the LexRO dimer dissociates, causing dissociation from the operator sequence, and initiates the expression of ccdB， eventually leading to cell death. We used LexRO, pHybrid 2)-114 version, SD7, ccdA, and ccdB to construct the regulation system and obtained the composite part<partinfo>BBa_K5136231</partinfo>, which was assembled on the expression vector pSB4A5.
  
 
<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/haoxihuan/and-gate.png" width="700px"></html></center>
 
<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/haoxihuan/and-gate.png" width="700px"></html></center>

Revision as of 11:15, 30 September 2024


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

In the darkness, LexRO dimerizes and binds to the cognate operator sequence to repress the activity of pColE408. Upon blue light exposure, the LexRO dimer dissociates, causing dissociation from the operator sequence, and initiates the expression of ccdB， eventually leading to cell death. We used LexRO, pHybrid 2)-114 version, SD7, ccdA, and ccdB to construct the regulation system and obtained the composite partBBa_K5136231, which was assembled on the expression vector pSB4A5.

Fig. 1 Gene circuits of verification system for hrp AND gate.


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.

Fig. 2 Gene circuits of hrp system regulated by the CspA CRE.

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