Recombinant Plasmid for Stress-Free Bacterial Curing
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Solution Overview
Problem
Current methods for curing endogenous plasmids from host cells are inefficient and often result in cell death due to the presence of Post-Segregational Killing systems, which can be toxic to the host cell upon plasmid displacement.
Innovation Solution
A recombinant displacement plasmid is introduced into the host cell, equipped with a nucleic acid molecule that neutralizes the toxic effects of the Post-Segregational Killing system and outcompetes the replication of the endogenous plasmid, ensuring host cell viability and efficient plasmid displacement.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If stress methods (high temperature, detergents, mutagens) are used to displace endogenous plasmids, then plasmid displacement efficiency is improved, but host cell mutation rate increases and phenotype alterations occur
Solution Approach 1:
The patent introduces a displacement plasmid as an intermediary carrier that mediates plasmid displacement through incompatibility mechanisms. This displacement plasmid contains a rep gene that produces Rep protein, which specifically targets and displaces the endogenous plasmid through replication competition, rather than using direct stress methods. The Rep protein acts as a mediator that achieves plasmid curing while maintaining host cell integrity and avoiding stress-induced mutations.
2Stability of the object's composition
If Post-Segregational Killing systems are present in endogenous plasmids, then plasmid stability is improved, but host cell death occurs upon plasmid displacement
Solution Approach 1:
The displacement plasmid is engineered to contain an antidote gene that is expressed before and during the plasmid displacement process. This preliminary action ensures that the antidote protein is present in the host cell to neutralize the toxin produced by the Post-Segregational Killing system, preventing host cell death. The antidote gene is strategically included in the displacement plasmid structure to provide protective action during the curing process.
3Productivity
If incompatibility-based displacement is used, then plasmid displacement efficiency is improved, but displacement of plasmids with unpredictable replication systems fails
Solution Approach 1:
The displacement plasmid is designed with modular parameters including different rep gene options (repA, repB, repC) that can be selected based on the specific endogenous plasmid target. The plasmid structure allows for adjustment of copy number control, origin of replication, and antibiotic resistance markers to optimize displacement efficiency for different plasmid types. This parameter flexibility enables the system to adapt to various plasmid replication systems while maintaining high displacement efficiency.
Data Source
AI summary
The present invention relates to plasmid curing, and particularly to efficient and stress-free methods for displacing resident or endogenous plasmids from a host cell, such as a bacterium. The invention extends to method of displacing a plasmid comprising a post-segregational killing system from a host cell, the method comprising introducing a recombinant nucleic acid molecule into a host cell harboring a plasmid comprising a post-segregational killing (PSK) system, characterized in that the recombinant nucleic acid molecule is adapted to neutralize the toxic effects of the plasmid's post-segregational killing system, and wherein the nucleic acid molecule is also adapted to outcompete or inhibit replication of the plasmid. The invention further extends to recombinant nucleic acid molecules that can be used in this method, as well as further uses of the methods and nucleic acid molecules of the invention.