To create our novel, translational-based biocontainment system, we accomplished several tasks. We first selected the N-carbobenzyloxy (CBZ) protecting group for leucine based on costs, experimental results, and modeling data. We then mutated the leucyl-tRNA synthetase using modeling software to create a mutant synthetase that confers complementarity to CBZ-leu but not normal leucine in the catalytic site and confers complementarity to normal leucine but not CBZ-leu in the editing site. A cleavage enzyme capable of cleaving CBZ-leu was chosen based on data in the literature and considerations on enzyme specificity for CBZ. The CBZ-cleavage enzyme from Nanduri, et. al. was cloned into a BioBrick and was shown to cleave CBZ-leu via observation of transformed leucine auxotrophic cells being able to grow on minimal media plates containing CBZ-leu.
There are a few more goals that we have in mind for our project in the future. First, in regard to the mutant leucyl-tRNA synthetase (m-leuS) gene, we frequently encountered pieces of DNA that went missing from our plasmid when we were cloning it. In the future, we would like to codon optimize the m-leuS gene to reduce the amount of homology between m-leuS and the wild-type leucyl-tRNA synthetase (leuS) gene in the genome. This will hopefully facilitate the cloning and use of this gene. Second, we would like to run a series of escapee propagation experiments in XL1-blue E. coli cells to assay the effectiveness of our biocontainment method. Escapee propagation would be tested in a variety of environments -- such as in water, in the soil, or in freezing cold temperatures -- to determine the versatility of our biocontainment method and test its limits. This will help researchers decide whether or not they can use synthetic translational control as a feasible biocontainment method for their genetically engineered organism in a targeted environment. Lastly, we would like to use CRISPR/Cas9 and lambda-red recombineering technology to simultaneously knock-out the wild-type leuS gene and knock-in the m-leuS gene and CBZ-cleavage enzyme into the genome. This provides greater stability of our biocontainment system since integration into the genome reduces the odds of horizontal gene transfer of the genes out of our organism when compared to keeping them on a plasmid. CRISPR/Cas9 also allows for efficiency and translatability of our biocontainment system between species for future implementation. These future developments will help take synthetic translational control one step further out of the proof-of-concept stage towards making it a reality.