We set out to create a device that would confer biocontainment on the translational level by modifying the cell to need a modified metabolite for survival. Translation is a key step in cellular growth and survival. Without the necessary metabolite needed to fuel our modified system, the cell is prevented from continuing translation.
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 from the protected leucine. Through research and experimentation, we determined that N-carbobenzyloxy-leucine (CBZ-leucine) fulfilled our criteria, and was also commercially available.
The cleavage enzyme will remove the CBZ group from the CBZ-leucyl-tRNA, allowing for normal leucine to be incorporated normally into polypeptides. The cleavage enzyme should cleave the CBZ group specifically and have minimal other downstream effects on cellular function.
Mutant Leucyl-tRNA Synthetase
tRNA synthetase enzymes serve an essential step in the polypeptide formation pathway: they join amino acids and tRNA molecules to form aminoacyl-tRNAs, which go on to the ribosome to form polypeptides. tRNA synthetases are amino acid-specific. In our project, we aim to replace the leucyl-tRNA synthetase with a mutated version that will charge tRNA with CBZ-leucine instead of wild-type leucine.
The mutant synthetase has two requirements: it should preferentially bind CBZ-leucine with tRNA, and also hydrolyze any wild-type leucine-tRNA that forms. The mutant has two active sites, one which creates the amino acyl-tRNA bond, and one which edits these amino-acyl-tRNA bonds. Thus our modeling aims to mutate both of these sites. The amino-acyl-tRNA bond formation active site should be sterically altered to select for CBZ-leucine, and the editing site should also be changed so that it recognizes wild-type-leucyl-tRNA as an incorrect pairing.
Implementation of the system
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.