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Expression System

The system we have designed relies on the presence of two Clickable Outer Membrane Proteins within the cells. To obtain cells which are functionalized with these proteins, we need an expression system capable of producing two proteins which carry non-natural amino acids. In addition to co-expression of two proteins, we thus need to gear up our bacteria with the tools to incorporate non-natural amino acids within their proteins. An overview of the expression system we used to obtain these bacteria is presented below.

Figure 1: As a proof of concept, we will construct and express our device within E.coli BL21DE3. The device we test features the fast cellular response signaling components. We will verify the construction of our device by conducting numerous experiments. These experiments include verification of the click reaction, of the individual signaling components, whether proximity invokes a response and if the aptamers work.
The BL21DE3 cells will be cotransformed with two plasmids. The first plasmid, pET-Duet1 (blue), carries the genes for the outer membrane proteins and expression is triggered by the addition of IPTG. The second plasmid, pEVOL pAzF (red), is necessary for the incorporation of the non-natural amino acid within the outer membrane proteins.

We enable our bacteria to incorporate the non-natural amino acid through the use of the Amber Stop Codon.