We wanted our interface to produce a protein that would inhibit the development of Staphylococcus aureus in the environment of the implant, and we wanted this peptide (RNA III inhibiting peptide) to be secreted from cytoplasm to the extracellular medium.

We decided to modify the Biobrick BBa_K237002 from iGEM SDU-Denmark 2009 Team. This Biobrick enables expression of RNAIII inhibiting peptide, we improved it by adding a secretion signal peptide to adress RIP to Escherichia coli Type II Secretion System and optimizing it for our chassis E. coli BL21 (DE3) pLysS strain.

Following the advice of Dr. Jean-Michel Betton, Research Director in the Structural Biology Department at the Institut Pasteur, we chose to try two different signal sequences to export our peptides, which he knew about and which are known to be efficient: MalE and DsbA. A secretion machinery called “secreton” then enables the release of proteins extracellularly. However, this system is not very well characterized yet, and it is a complex machinery composed of more than 10 proteins, so we did not plan to use it. However, since RIP is only a 7 amino acid protein, leaky release from the periplasm to the medium should be enough to obtain RIP in the medium [6].

Figure 1: Schematic diagram of BBa_K2616001