Maximizing protein expression in bacterial systems requires a deep understanding of the regulatory mechanisms influencing mRNA expression and protein yield. This project focuses on optimizing protein expression by manipulating the lac operon system in E. coli and investigating the impact of various factors on protein stability and yield. The research primarily centers on the MutT4 enzyme from M. smegmatis, which was genetically modified to include a translational ramp sequence aimed at enhancing protein stability. Despite successful initial PCR reactions and Gibson Assembly, attempts to integrate the translational ramp sequence into the MutT4 plasmid were ultimately unsuccessful. However, optimal conditions for protein expression were determined through experiments involving temperature and IPTG concentration variations. The results indicated that a temperature of 28°C and IPTG concentration of 0.5 mM yielded the highest levels of MutT4 enzyme. Further attempts to improve expression through environmental factors such as metal-ion cofactors and pH adjustments were inconclusive. This project highlights the importance of optimizing both genetic constructs and environmental conditions to achieve high levels of protein expression in bacterial systems.

• This report represents the work of one or more WPI undergraduate students submitted to the faculty as evidence of completion of a degree requirement. WPI routinely publishes these reports on its website without editorial or peer review.

Creator

• Cochran, Matt

Publisher

• Worcester Polytechnic Institute

Identifier

• E-project-081224-135628
• 124009

Advisor

• Bafaro, Elizabeth
• Roberts, Louis Anthony

Year

• 2024

Date created

• 2024-08-12

Resource type

• Major Qualifying Project

Major

• Biochemistry

Source

• E-project-081224-135628

Rights statement

• In Copyright

Last modified

• 2024-09-19