MEMBRANE PROTEIN EXPRESSION OF ENGINEERED ESCHERICHIA COLI STRAINS AFTER TRANSFORMATION

Background:The performance of bioengineered strains of bacteria is integral to the companies that depend on these strains to produce products. These products can range from biofuel to medication. The Joint BioEnergy Institute (JBEI) in cooperation with Lawrence Berkeley National Laboratory has set out to explore and improve the performance of bioengineered bacteria. One key component of the performance metric is membrane capacitance, which measures how readily a bacterial strain is able to utilize modified metabolic pathways to express additional membrane proteins. Methods: JBEI researchers selected Escherichia colistrains used to produce biofuels and then subjected them to two distinct assays to measure their membrane capacitance. Both of these assays were conducted with varying levels of Isopropyl β-D-1-thiogalactopyranoside (IPTG) as an inducing agent. These assays consisted of first using a Tecan 200 liquid plate handler and second a Fluorescence Activated Cell Sorter (FACS). One assay measured expression of fluorescent tagged membrane proteins over a 24-hour period using a Tecan 200 liquid plate handler. We also used a FACS assay to measure the expression of fluorescent proteins in each individual cell after incubating 24 hours. Results: We found that strains of E. colithat had been transformed with multiple bioengineered plasmids had marked decreases in fluorescent protein expression when compared to strains transformed with one bioengineered plasmid. A strain containing only a single plasmid for Green Fluorescent Protein (GFP) production had a mean fluorescence 152% higher than the next closest double plasmid strain. All strains containing a single plasmid for Red Fluorescent Protein, mCherry, had a mean fluorescence at least 250% greater than the next closest double plasmid strain. These results were still upheld when the bioengineered strains made use of high-copy Origins of Replication and with varying levels of IPTG induction. Conclusion: The data suggest that addition of successive plasmids to a target strain impedes the ability of that strain to maximally produce membrane proteins, which may impact industrial biofuel applications. Whether the relationship between number of plasmids and membrane protein production is a linear or exponential is unclear. We postulate that additional transformations (e.g., triple, quadruple, etc.) with bioengineered plasmids will result in even lower levels of membrane protein production of the transformed bacterial strain.