**2.4 Conclusion**

In this chapter, we demonstrated the high-throughput of cell-free protein synthesis in enzyme kinetic studies. We also explored the possibility of using UAA to enhance OPH substrate binding by testing a method utilizing GCE machinery to incorporate selected UAAs into OPH. A TAG stop codon was inserted into OPH to replace these sites, and OPH mutants with UAA substitution were expressed successfully. A total of eleven UAA substitutions were generated, with 3-methyl-histine substitutions identified as the most suitable to replace the OPH active site histidine network. Results of kinetic studies of these mutants show significantly improved OPH substrate binding affinity.
