Plasmid Copy Number Regulation via Temperature-Sensitive Replication
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Solution Overview
Problem
Current methods for controlling plasmid copy numbers in prokaryotic hosts are laborious and often result in uncontrolled loss of target gene expression cassettes, metabolic burdens on the host, and unreliable plasmid integration into the host chromosome, especially under stressful conditions.
Innovation Solution
A method involving a prokaryotic host with a plasmid having an origin of replication activatable by a plasmid replication initiator protein (Rep), where the expression of the Rep protein is regulated to adjust the plasmid copy number using heterologous promoters and repressors, allowing for controlled plasmid replication and integration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If plasmid copy number is increased to enhance target gene expression, then gene dosage increases, but metabolic burden on host increases and host viability decreases
Solution Approach 1:
The invention employs a temperature-sensitive origin of replication that allows dynamic control of plasmid copy number. At permissive temperatures (e.g., 30°C), the origin functions normally allowing plasmid replication. At restrictive temperatures (e.g., 42°C), replication is blocked, enabling the system to adapt copy number to metabolic conditions and reduce burden when needed.
Solution Approach 2:
The invention changes the temperature parameter to control plasmid replication. By shifting from permissive to restrictive temperature, the system transitions from allowing replication to blocking it, thereby controlling copy number and metabolic burden without requiring additional genetic components.
2Quantity of substance
If temperature-sensitive origin of replication is used to restrict plasmid replication, then plasmid copy number can be controlled, but plasmid loss occurs at growth stages where low copy number is desired
Solution Approach 1:
The invention introduces a mediator plasmid that carries the rep gene and a selectable marker. This mediator serves as an intermediary between the temperature-sensitive plasmid and the host, providing continuous selection pressure to maintain the plasmid even when replication is blocked at restrictive temperatures, thereby preventing plasmid loss.
Solution Approach 2:
The system employs feedback through selectable markers that provide continuous selection pressure. When plasmid copy number drops below a threshold or plasmid is lost, the selectable marker phenotype is lost, allowing selection against plasmid-free cells and feedback maintenance of plasmid presence in the population.
3Object-affected harmful factors
If plasmid replication is completely blocked to achieve low copy number, then metabolic burden is reduced, but integration efficiency into host chromosome decreases
Solution Approach 1:
The invention uses periodic temperature shifts to alternately permit and block plasmid replication. During integration phases, the temperature is shifted to restrictive conditions to block replication and reduce metabolic burden. During expression phases, temperature is returned to permissive conditions to allow replication and increase copy number for higher gene dosage.
Data Source
AI summary
Described herein are materials and methods for user-controlled adjustment of plasmid copy numbers.