In the Design section, we designed StarCore sequences as compound BioBricks fusing an AMP sequence to a multimeric core. In this section, we use cell-free expression to produce StarCore proteins.

Cell-free expression allows us to bypass the toxic effect that our living cells would have if they produced our StarCore for hours but also allows us to have high throughput expression and to quickly screen many compounds.

StarCore fusion proteins were expressed using the myTXTL Sigma 70 Master Mix Kit, generously provided by our team sponsor, Arbor Biosciences. As an expression vector, we used pACYCDuet-1 from Novagen. This vector is widely used for protein production in strains of E. coli that express T7 polymerase such as BL21 (DE3). It contains a T7 promoter upstream of a strong RBS and a lac operator, allowing IPTG-controllable protein expression.

To express from the T7 promoter, it was necessary to first produce T7 polymerase in the cell-free extract. For this purpose, we used the plasmid P70a-T7rnap, supplied by the manufacturer. We also included 100 uM IPTG in the master mix, to relieve lac repression.