Team:WHU-China/Proof Of Concept
This year we didn’t achieve a comprehensive proof-of-concept but a basic one via several fundamental experiments, however, we did design the subsequent experiments and prototypes to demonstrate that our negotiator probiotics are likely to function in a relevant context. The feasible Quenching Module and Sensing Module are decisive factors in our proof-of-concept work. In Engineering Success page, the DBTL cycles on these two modules are described, and next we will illustrate how we connected two constructed modules with hardwares to achieve a basic proof-of-concept.
Our DIY co-culture device has thorough documentations in page
Hardware including a list of purchasable
components, an operation protocol and teaching
videos. In our plan, it serves as a experimental platform to perform negotiator-criminal co-culture
and biofilm assays, for demonstrating Quenching Module.
Since no significant progress is made in Quenching Module construction, we performed the co-culture
experiment of E. coli Nissle 1917, a natural strain instead of the engineered version, and P.
aeruginosa, which turned to be successful. Based on the expected co-culture results, the future
plans to achieve an advanced proof-of-concept are proposed. After in vitro enzymatic assays to
select optimal quorum quenching enzymes, the engineered EcN will be co-cultured with P. aeruginosa
in the device. Quorum sensing biomarkers of P. aeruginosa (such as QS-regulated GFP expression in
engineered P. aeruginosa, and biofilm assays) can be measured to quantify the function of Quenching
Module in a relevant context.
Figure 1: co-culture of E. coli Nissle 1917 and P. aeruginosa.
Importantly, to demonstrate Quenching Module in silico, a gro model concerning spatial factors to simulate the process of quorum quenching was established. The results proved our idea from an alternative perspective, and the analysis can be found in the page of Model.
Figure 2: gro simulation of quorum quenching.
This year, we performed Transwell tests to quantify the chemotaxis of immune cells. Coupling with flow cytometry, the promising results helped us select optimal chemokines, which are shown in Engineering Success and Results. However, due to the defeat in plasmids construction, the recombinant chemokines expressed by E. coli cannot be tested in Transwell tests. We envision the functions of recombinant chemokines tested next year.
Figure 3: flow cytometry to select the optimal chemokines.
Also, the system of signal reception, amplification and control, isn’t constructed because of failures in molecular cloning. Next year, after constructing the complete Sensing Module with well-selected chemokines, proof-of-concept experiments will be performed upon a microfluidic chip that simulates human lower respiratory tract. As for future proof-of-concept work, microfluidics is considered one of the most important platforms in our project, because of its ability to mimick the practical environment where negotiators (engineered probiotics) interact with police squads (immune cells). The microfluidic platform is also applicable for a P. aeruginosa infection model, thus offering us opportunities to comprehensively investigate criminal-negotiator-police squad relationships.
Figure 4: microfluidic chip as the optimal platform for a final proof-of-concept.
In summary, we achieved a basic proof-of-concept this year and drew a blueprint for further demonstration efforts. Hopefully, a more complete proof-of-concept will be accomplished in phase II of our project.
