Modular plasmid design for autonomous multi-protein expression in Escherichia coli

Area of Science:

• Synthetic biology
• Molecular biology
• Genetic engineering

Background:

• Molecular and synthetic biology tools facilitate the creation of novel biological systems, including genetically engineered microorganisms and recombinant proteins.
• Efficient development of tailored biological solutions requires effective strategies for designing genetic circuits.
• A need exists for independent and controllable systems for the co-expression of multiple genes.

Purpose of the Study:

• To characterize a set of bacterial plasmids for recombinant expression in Escherichia coli.
• To enable the independent and controllable co-expression of up to three genes from a single plasmid.
• To demonstrate the utility of the system for producing complex biocatalysts.

Main Methods:

• Development of bacterial plasmids featuring four common expression cassettes: RhaS/RhaBAD, LacI/Trc, AraC/AraBAD, and XylS/Pm.
• Utilized Golden Standard Molecular Cloning kit assembly for creating three-gene monocistronic expression systems.
• Validated the system by expressing a triple-enzyme cascade for biocatalyst production.

Main Results:

• Characterized individual expression cassettes for strengths and limitations.
• Demonstrated independent induction and autonomous expression of up to three recombinant proteins from one plasmid.
• Successfully produced a complex triple-enzyme cascade, confirming system applicability for biocatalyst production.

Conclusions:

• The developed strategy offers a valuable approach for multigene expression, complementing existing co-expression methods.
• Thorough characterization provides insights into cassette performance and potential limitations for future research.
• Understanding defined cross-talks enhances knowledge of metabolic mechanisms affecting heterologous gene expression in bacterial hosts.