Team:Manchester/Experiments
EXPERIMENTS
Our initial design idea was to detect a food pathogen with a biosensor that was integrated into the food that it was protecting. We considered using many different strains for our biosensor, our primary idea being to use an edible E. coli strain (Nissle 1917). E. coli Nissle 1917 is well-known as being a probiotic strain and has been marketed as such. However, after lots of consultation (link to HP), our idea was refined: to use a strain native to cheese (Lactococcus lactis) that could detect a specific Listeria monocytogenes quorum signal (auto-inducing peptide; AIP), and would then alert the consumer to food contamination by changing colour.
This led to the design of two main circuits (link to parts): a detection circuit, which would detect the quorum signal and change colour, and a testing circuit, which would produce the AIP and allow us to test the detection circuit without having to work with the pathogenic L. monocytogenes. Ultimately, in our designs, we split the detection circuit into two devices as the whole circuit was too large to synthesise. One device (AgrA+C) produces the receptor histidine kinase and response regulator needed to detect the AIP and the other (PII) has a promoter that the response regulator binds to drive transcription and a reporter gene. We initially chose amilCP as our reporter gene as it is well-characterised in iGEM and produces a strong blue-purple pigment within 24 hours (in E. coli). Further investigation later led to the Glasgow iGEM 2016 wiki which suggested that amilCP may be unsuitable for expression in lactic acid bacteria due to acidic cellular conditions. In later constructs, we, therefore, used gFasPurple as our reporter gene as it was more representative of the colour we wanted to produce and did not have any known negative characteristics for our biosensor. We designed these circuits to be expressed in E. coli and L. lactis due to anticipating difficulties with an expression of membrane proteins, as well the ease of use of E. coli for DNA assembly.
Because it was important for our circuits to work in both E. coli and L. lactis, we needed promoters that would function well in both chassis. We found a library of such promoters in Jensen and Hammer’s 1998 paper (link) and whilst they are already quantified, we wanted to try quantification with chromoproteins, both to see if this is a valid method of quantification and to gather more data for the model(link). To this end, we designed two devices using these promoters; P15 and P25, and each consists of the related CP promoter (so CP15 for P15), a RBS (BBa_B0034), gfaspurple and terminators (BBa_B0010 and BBa_0012).
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