Adaptive Laboratory Evolution and Recombination Efficiency of the Genderless E. Coli Strain for Improved Tolerance of Industrially Relevant Stressors
Amy Nguyen, Joshua Luu-Nguyen, and Vivian Nguyen
Advisor: Dr. Katy Kao (SJSU)
Embedded Files
Adaptive laboratory evolution (ALE) is a powerful tool for enhancing microbial performance under specific environmental conditions and is applied in bioengineering to create industrially relevant microbial strains. This project focuses on improving the metabolic adaptability and genetic robustness of a novel E. coli Genderless+ strain, a genetically engineered variant capable of undergoing sexual recombination to accelerate evolutionary adaptation. Targeting non-preferred carbon sources—acetate and xylose—commonly derived from lignocellulosic biomass hydrolysates, we aim to enable this strain to thrive on substrates that are traditionally inhibitory to E. coli growth.
ALE experiments are performed by serial batch culturing under gradually increasing concentrations of acetate and xylose, applying selective pressure to promote beneficial mutations. Growth kinetics and phenotypic assays are used to monitor adaptation, and evolved populations are screened to isolate high-performing mutants.
To complement evolutionary adaptation studies, we also designed a method to assess the recombination efficiency of the Genderless+ strain. Using CRISPR/Cas9 gene editing, six pseudogenes varying in distance from the origin of transfer were tagged with fluorescent markers (GFP or RFP), enabling quantitative measurement of recombination events under various environmental conditions, including carbon source variations and elevated temperatures. These experiments provide critical insight into the genetic stability and adaptability of the Genderless+ strain, informing its broader application in synthetic biology and biomanufacturing.
Together, these studies integrate adaptive evolution and recombination analysis to develop a robust, engineered E. coli platform capable of efficient carbon utilization and enhanced genetic adaptability, paving the way for sustainable bioproduction using renewable feedstocks.
Page updated
Report abuse