Difference between revisions of "Team:Virginia/ProjectDesign"

Synthetic biologists struggle to prevent the proliferation of genetically engineered organisms (GEOs) in natural systems. Containment methods that operate in ecological settings must provide security comparable to physical containment. Current methods fail to effectively inhibit horizontal gene transfer and environmental supplementation, and impose evolutionary pressure through the propagation of spontaneous revertants. Synthetic Translational Control (STC) currently utilizes a redesigned leucyl-tRNA synthetase and cleavage enzyme in an E. coli chassis to confer metabolic dependence on a synthetically modified leucine capable of conversion to L-leucine. Due to the semi-semantic property of this device, organisms cannot metabolically bypass our constraints using environmental supplementation and will display greater resistance to evolutionary escape relative to traditional synthetic auxotrophs. Our work provides advancement in biosafety by isolating GEOs from the environment via a reliance on modified metabolites. STC will become a benchmark for biocontainment devices and will allow for countless new applications in synthetic biology.

To create an effective biocontainment system, when implemented into a target cell, our system must restrict the cell in such a way that it only grows and replicates when a non-natural metabolite is present. Cells using this system will be unable to survive outside of controlled environments, reducing the risk of escape. To accomplish this goal, we aimed to select a protecting group for leucine and a corresponding cleavage enzyme to remove the protecting group, and to replace the wild-type leucyl-tRNA synthetase with a mutant leucyl-tRNA synthetase of our design.

The biocontainment functionality of this system begins at metabolite uptake. The cell must be able to take up a non-natural metabolite that is not typically readily available in the environment. We needed to select an essential metabolite to the cell so that without that metabolite, the cell would die. Amino acids are key precursors to all proteins. We chose to modify the amino acid leucine. The non-natural modified leucine, after uptake, would then be charged with tRNA molecule to become a modified-leucine-tRNA molecule via a synthetase enzyme. However, this step requires a modified synthetase so that the modified leucine, rather than wild-type leucine, is preferentially charged with tRNA.

Next, the charged protected-leucyl-tRNA molecule must have its protecting group removed. Once its protecting group is removed, the wild-type leucyl-tRNA can continue on to polypeptide formation. In this way, the cell is incorporating normal leucine amino acids into proteins, while still being reliant on protected leucine.

Our protecting group needs to bind to the N-terminus of leucine, as the C-terminus of the amino acid has to bind to tRNA. Another essential requirement is that the organism, in our case E. coli, must be able to uptake the protected leucine molecule. The protecting group also needs to be large enough to be sterically distinct from wild-type leucine. Thus, the mutant leucyl-tRNA synthetase that we design will be able to enzymatically distinguish the wild-type leucine and the protected CBZ-leucine. Through research and experimentation, we determined that N-carbobenzyloxy-leucine (CBZ-leucine) fulfilled our criteria.

The cleavage enzyme will remove the CBZ group from the CBZ-leucyl-tRNA, allowing for normal leucine to be incorporated normally into polypeptides.

This synthetase, designed using our protein modeling software MUT, is designed to preferentially bind CBZ-leucine with tRNA, and hydrolyze any wild-type leucine-tRNA that forms.

To implement this system completely, the genes for both the CBZ cleavage enzyme and for the mutant leucyl-tRNA synthetase need to be readily transcribed and translated into functional proteins in the organism. Additionally, to ensure that leucine is not usable in the organism's metabolism, the wild-type leucyl-tRNA synthetase gene needs to be knocked out. In this way, we would guarantee that the organism cannot survive without supplementation of the CBZ leucine.