Understanding the Lifetime and Rate of Protein Production in Cell-Free Reactions While Maximizing Energy Use

Citation

Roychoudhury, Ankita (2021) Understanding the Lifetime and Rate of Protein Production in Cell-Free Reactions While Maximizing Energy Use. Senior thesis (Minor), California Institute of Technology. doi:10.7907/94ms-b419. https://resolver.caltech.edu/CaltechTHESIS:06052021-045112334

Abstract

Liposomes, or vesicles, offer promising applications in fields including biofuel synthesis, drug delivery, and toxin removal. Programmable liposomes can be used for optimal protein synthesis and to prototype genetic technologies. However, one of the major challenges is the short lifetime of protein production. Here, we add metabolites and molecules to cell-free reactions at different times to interrogate their importance. Through this testing, we find that ATP only slightly enhances protein synthesis, and ADP can help a reaction reach steady state faster. We also note that the excess of particular molecules, such as NAD and 3PGA, can halt protein production. With this data, we developed a more accurate chemical reaction network-based model for cell-free reactions. We also begin to study an unexplained discrepancy in protein production between bulk and vesicle dynamics. To quantify protein synthesis, we use E. coli extract and energy buffer, often called cell-free transcription and translation (TXTL), with a chosen DNA template both within vesicles (encapsulated) and without (bulk). We have also been able to uncover fundamental properties of transcription/translation systems. We supplement this data with computational models utilizing chemical reaction networks. We established a vesicle setup with membrane pores and supplemental energy buffer on the outside which increased the efficiency of protein synthesis. By using chemical reaction network models, we have highlighted differences and similarities between models and experiments. With this setup, vesicles can be used for more complicated applications, such as drug delivery or genetic construct testing.

Thesis Files