Part:BBa_K639003:Experience

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Imperial iGEM 2013

We intended on using BBa_K639003 to detect whether our cells were stressed when grown in mixed waste, mixed waste conditioned media or in the presence of plastic toxic byproducts. However, as the data below shows, this mCherry biosensor is very leaky, with clear expression in an un-induced or non-stressed state. However based upon our characterisation data the biosensor does induce increased mCherry expression as expected and therefore we recommend future teams try to improve the part by reducing its leakyness.

We did however find the sensor to be useful to our project as a marker of our chassis in extended waste toxicity assays. The data is included below.

Cell growth over 6h with IPTG induction. mCherry production is induced by the stress pathway and detection of ppGpps. In order to bypass this, we induced with IPTG which inhibits LacI, resulting in mCherry expression.

Fluorescence of the cells under IPTG induction over a 6h period.

BBa_K639003 transformed into E coli. strain MG1655. Pink colonies are visible, which relate to 'leaky' RFP production

BBa_K639003 transformed into E coli. strain MG1655. Cells were grown at 37oC in 4ml LB with 0, 1 or 2mM IPTG. At 6 hours post IPTG induction, cells were spun down.