One of the most important parts of science has just not always been to break boundaries and novelty in science but to use the knowledge learned from the research and use it for the common good. Not only as an interesting science project, we believe that our project can also be used in exciting applications in the real world. Here we propose some ways in which our project could help solve pressing issues or act as a stepping stone in the area of scientific research. It is important to identify potential risks, hazards, and other obstacles that might hinder implementation. For that, we have identified our end-users, and designed strategies to mitigate potential risks.
National enzyme producers
Researchers working in the area of protein interactions
Researchers working in the area of recombinant proteins expression
Researchers working in the area of drug and vaccine delivery
National enzyme producers still rely on wild-type strains to produce their products, without any genetic modifications or engineering done. This leads to only a few choices of enzymes produced that are available. The lack of modifications also leads to the enzyme produced to be diluted within the media of the bioreactor in which this problem needs to be solved in the downstream process. The downstream processes that are currently used by national producers are ultrafiltration and microfiltration, which are expensive and require process and time.
We are planning to insert our system into a hypervesiculating strain, that is the JW0729-3, an E. coli with a TolA deletion. This strain has been proven to induce the vesiculation mechanism of E. coli which along with our project using Outer Membrane Protein, could increase the protein yield. The increase of protein yield has also been proven by Henry et al. in 2004 , stating that deletion in any of the gene within the Tol-Pal system (tolA, tolQ, tolR, tolB, and pal) is causing an increase in vesiculation.
As vesicles would sometimes be produced in a very low yield, one of our ideas for future improvements including integrating our system in a hypervesiculating strain of Gram-negative bacteria in order to increase the vesicle yield while also increasing the amount of surface proteins on the vesicles, as this would make the efficiency for OMV as drug or vaccine delivery even better.
It has been proven by several papers that the deletion in the tolA-Pal system leads to an increase in vesiculation [1,2,3]. But due to the time limit we have and the lack of response from the centre we are planning to buy the strain from, we were unable to test our system using the strains with said deletions.
Currently in our system design, each eCPX will bind with the linker and amilCP only on the N-terminal of eCPX. We acknowledge that eCPX have both of its N- and C- terminals to be on the same orientation, that is extracellularly . Thus, this kind of advantage that eCPX has would give an opportunity for researchers in the future to explore the ability of eCPX to hold two amilCP or another Protein of Interest both in its N- and C-terminus at once. From this, the amount of protein or enzyme production will increase twice than using another type of outer membrane proteins or integral proteins that resides in the outer membrane, aside from eCPX.