Prototyping of Genetic Parts through Clostridia-Based Cell-Free Platforms

Climate change is a multi-faceted and growing global crisis that is exacerbated by greenhouse gas emissions such as CO2 and CO. A large source of such pollution is the release of waste gas streams from industrial scale productions such as coal-powered plants and steel manufacturing. Such emissions negatively impact the environment and methods to reduce pollution must be leveraged across industry. Biotechnology and strain engineering have been rising sectors in recent decades as microbes hold great potential for undergoing complex chemical reactions, however, engineering organisms is an extremely lengthy and costly process from the growth of cell cultures to arduous genetic modifications. These slow time frames result in long periods of research for new technological strategies as well as for any large-scale implementation. The utilization of cell-free systems and cell-free protein synthesis (CFPS) can greatly accelerate research timelines by producing proteins and mimicking metabolic pathways in vitro. This allows for rapid experimentation and optimization of genetic parts, mutations, and enzymatic pathways prior to in vivo strain growth, with potential for high throughput screening strategies as well. I applied this framework of cell-free prototyping towards Clostridia strains: anaerobic bacteria with unique metabolic pathways that are capable of converting CO2 to short chain organics compounds. As this bacterium has just recently begun to enter commercial plants, its cell-free platform and distinctive pathways are much less studied in comparison to other microbes such as E. coli. I revealed ranges of CFPS efficiency and demonstrated genetic protein product control through successful modification and replacement of key genetic parts such as promoters, terminators, and ribosomal binding sites. This work provides insights and expands our knowledge into the genetic control of Clostridia cell-free systems, multi-step synthesis development, and progresses towards sustainably circular environments.