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Team:Amsterdam/project/molecular design
From 2012.igem.org
Sensor
What does a sensor mean to the Cellular Logbook?
A logbook aims to store the occurrence, encounter or presence of an external signal. Whether this signal comes from a metabolite, a chemical or a toxin, compound or substrate, does not matter! What matters for our Cellular Logbook is if the signal can be taken up by the cell and is able to activate a promoter that will induce the transcription of our writer module. Therefore any well-characterized promoter in the parts registry can be used in the sensor module.
For our proof-of-concept we chose to implement two different promoters as sensors:
Lac-hybrid promoter:
The lac promoter and its different protein components have been studied for decades and is widely used as one of the common systems for recombinant protein production in E. coli.
In its repressive state, the LacI repressor, an allosteric protein constitutively expressed by E. Coli, binds the promoter with high affinity, thereby preventing transcription. Once bound by an inducer such as lactose or IPTG, the repressor is released from the promoter and the RNA polymerase complex can be formed to enable synthesis.
Our first sensor uses the lac-hybrid promoter (BBa_R0011) together with a medium ribosomal binding site (BBa_B0032). Derived from the original Lac Operon (BBa_R0010), this adaptation does not rely on the presence of glucose, but only on lactose.
Arabinose promoter:
This promoter is derived from wild-type E. coli and has a modified AraI1 site, which causes this promoter to be less responsive to low concentrations of induction and therefore exhibits a lower maximum response.
pBAD is very specifically activated by L-Arabinose. In the absence of arabinose, the repressor protein AraC binds to AraI1 AraO2, blocking transcription. In the presence of arabinose, AraC binds to it and changes its conformation such that it interacts with the AraI1 and AraI2 operator sites, allowing transcription.
For the characterization of our system, we intentionally chose a weak version of the promoter, the pBAD-weak (BBa_K206001), from the parts registry.
