Difference between revisions of "Team:Virginia/Implementation"

Beyond a proof of concept to determine the success of Manifold, further tests will be required to determine the effectiveness of our design and its scalability. The primary test for efficacy will be an HPLC analysis of the culture broth in order to identify the concentration of resveratrol being produced. First two standard curves will be made based on known concentrations of resveratrol and 4-coumaric acid in a 10-fold dilution of the culture medium. This will also allow for a background to be established. Next, Manifold containing DH10β E. coli will be cultured in the presence of 4-coumaric acid over the course of two days and samples of the culture broth will be taken every six hours. Ten-fold dilutions of these samples will then be run through an HPLC machine and the concentrations of resveratrol and 4-coumaric acid will be assessed. In addition a strain of DH10β E. coli modified to produce only 4CL and STS will also be cultured in the same 4-coumaric acid containing medium, and samples will also be taken on 6 hour intervals for two days. This will serve as a positive control, while a third culture of untransformed DH10β E. coli will serve as a negative control. The HPLC data from each culture will be compared to the standard curves so that the concentrations of resveratrol and 4-coumaric acid can be plotted over time. To see if the Manifold system adequately increases the flux through the pathway, the final ratios of resveratrol to 4-coumaric acid can be compared. If there is no flux leakage, then this ratio should be equal to one. In order to see if an increase in overall product is observed, the ratio of resveratrol concentrations between the experimental group and control groups can be used. Finally, to see if the rate of resveratrol is increased by the system, a linear regression can be fit to each concentration vs time plot for resveratrol and the slopes can be compared. If our yields support the hypothesis that Manifold nearly eliminates flux leakage, we have the data required to move on to testing other enzyme pathways.

If our implementation for Manifold is successful with resveratrol, we can move onto testing other enzyme pathways in similar fashions. Validating Manifold with pathways that are applicable to more important pharmaceutical pathways will be our priority, and we have explored options relating to statins, medications meant to reduce cholesterol levels, in order to extend our system to the production of more diverse compounds. Doing so will demonstrate the scalability of Manifold, and show that our platform technology in the space of biologics has the potential to increase yields and bolster efficiency.

Manifold represents a truly exciting advance in the field of synthetic biology. By channeling metabolic flux through BMCs and DNA scaffolds, Manifold fixes and confines metabolic pathways in space, allowing for dramatic increases in flux through the pathway. By localizing enzymes and substrates together within BMCs, required interactions happen more frequently. Additionally, by compartmentalizing the pathway within BMCs, reactions and intermediates are isolated from the rest of the cell which is both convenient for the researchers and the microbe. It’s difficult to understate the impact Manifold will have on the field of synthetic biology and pharmacology. If successful, it will revolutionize how bacteria are engineered to synthesize anything from bulk chemicals to pharmaceuticals to biofuels. The implications on the pharmaceutical world alone are astounding. The cost of production of virtually any drug synthesized in bacteria can be reduced, in turn, reducing the cost of life-saving as well as everyday drugs. While creating our proof of concept design for resveratrol, we focussed mainly on the pharmaceutical industry, determining how the synthesis of this supplement will impact the surrounding field, but if the use of Manifold is successful, any form of enzymatic compound production has the potential to be heightened. Through the combination of BMCs and DNA scaffolds to channel metabolic flux, Manifold represents an exciting advance in the field that cannot be underestimated. Though there is much work to be done yet, the team is thrilled to continue crafting its approach to Manifold.

There arise a number of safety and ethical considerations that will require close attention. As pointed out before, who should first benefit from our technology? Are there ways in which Manifold can be manipulated for malicious purposes? These are difficult questions and there don’t appear to be any simple answers just yet. This is exactly why it’s so important to create a discussion within our group and with the community around us so the ways in which this technology will disrupt the field of synthetic biology will be better understood. Foundational advances are powerful tools; they derive much of their impact and excitement from the possibility to adapt the advance to an array of purposes, organisms, and disciplines. Unfortunately, there exist perhaps an equal number of ways to commandeer the technology for selfish or malicious motivations as there are ways to use the technology for well-meaning adaptations. Here lies the worst case scenario regarding Manifold. In the wrong hands, this technology can be used to increase production of compounds to be used as weapons. Fortunately, with the high metabolic load placed on our engineered E. coli cells, it follows that our organism will face a significant disadvantage if released into the environment. For this reason, there is no reason to suspect that the engineered organism cause for concern.