Anthony Eckdahl, Kyla Roland, Elise Edmond, Tatianna Travieso, Caroline Willis, Maria Roja
sFaculty Sponsor: A. Malcolm Campbell, Laurie Heyer
Our undergraduate researchers are interested in advancing the field of metabolic engineering for the production of useful metabolites that serve as biofuels, pharmaceuticals, or industrial chemical feedstocks. We published an approach to the metabolic engineering of bacteria called programmed evolution, which introduces variation in regulatory elements for the production of enzymes that control orthogonal metabolic pathways. Programmed evolution uses in vivo fitness modules that enable selection by employing riboswitches to transduce the production of desired metabolites into antibiotic resistance. Because of limitations imposed by in vivo programmed evolution such as crosstalk between orthogonal and native bacterial metabolism, the toxicity of some metabolites, and poor representation of large variation spaces, we want to reinvent programmed evolution as an in vitro strategy. This preliminary report describes our first steps of comparing lysates from two different strains of E. coli and using them to carry out cell-free protein synthesis (CFPS). We also designed, constructed, and have begun testing a fitness module based on the expression of the restriction enzyme SmaI. We are working toward a proof-of-concept system in which combinations of DNA regulatory elements that enhance the production of caffeine demethylase are selected for their ability to produce theophylline. Theophylline production will turn on a riboswitch that leads to a burst of SmaI expression. Localized SmaI activity is expected to preferentially cleave successful control elements over unsuccessful ones, allowing purification and identification of previously unknown requirements for CFPS-controlled metabolism. The successful development of in vitro programmed evolution would enable researchers to exploit the power of selection to discover combinations of control elements for the optimal yield of metabolites that are not easily produced in vivo.