Team:NTNU-Trondheim

All gram negative bacteria produce outer membrane vesicles (OMVs) by bulging of from their outer membrane. These OMVs contain different proteins and carry out varies functions such as quorum sensing, involvement in pathogenesis and transporting enzymes to distal locations. In our project we wish to prove that OMVs can be manipulated and to thereby implicate OMVs potential as a drug delivery vehicle. This is an innovative and novel approach to create a safe way to deliver drugs in the body.

We are going to prove that the OMVs can be manipulated by introducing two different proteins. The first one is a fusion protein of GFP and RFP. This will make it possible to visualize the vesicles as well as investigating the new functionalities of fusing two fluorescent proteins together. The second protein we wish to introduce to the vesicles is the transmembrane protein G derived from Streptococcus dysgalactiae ssp. equisimilisi. Protein G is known to bind to Human Serum Albumin (HSA) which helps S.dysgalactiae subsp. equisimilis hide from the immune system. Protein G will therefore be a potential important piece in a drug carrier by masking it from immunological destruction and making it stable in the blood stream.

In order to direct the proteins into the periplasm and vesicles they need to have a tat signal peptide at the N-terminal. This will transport the proteins through twin-arginine translocation pathway (Tat pathway). Studies show that a twin-arginine signal peptide is able to direct the export of active green fluorescent protein (GFP) in Escherichia coli . We also wish to be able to regulate the production and thereby the export of proteins to OMVs. In order to accomplish this task we will set the gene constructs under regulation of the Pm/Xyls promotor system. This is a positive regulation system that is activated by the inducer m-toluic acid

Introducing these proteins into vesicles also demonstrate that it is indeed possible to manipulate the content and therefore the properties of OMV's. We will prove the concept that OMVs can be engineered to meet the criteria of a drug delivery vehicle.