Variant inc RNA and R47C rpoC for Plasmid Copy Control

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Current biotechnological applications face challenges in stably incorporating target genes into microorganisms and controlling the yield of protein products, as techniques for replicating plasmids and expressing genes often lead to metabolic burdens, plasmid loss, and unstable expression levels, with no general approaches to predictably modify plasmid copy number or RNA polymerase β′ subunit to achieve desired outcomes.

Innovation Solution

A nucleic acid molecule comprising a variant inc coding strand that encodes a variant inc RNA, which regulates plasmid copy number, and a recombinant microorganism with a variant rpoC RNA-polymerase β′ subunit protein coding sequence that includes an R47C substitution, allowing for controlled plasmid copy number modulation without affecting chromosomal copy numbers.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If plasmid replication and gene expression techniques are used to increase target gene yield, then productivity is improved, but plasmid stability and metabolic burden worsen

Engineering Contradiction:

Improvetarget gene yieldVSAvoidplasmid stability

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent modifies the inc RNA coding sequence by introducing specific nucleotide substitutions (e.g., C to U at position 60) to change the regulatory parameters of plasmid replication. This alters the copy number control mechanism, enabling increased target gene expression while maintaining plasmid stability through controlled replication parameters rather than uncontrolled metabolic burden.

Inventive Principle:

Principle #35Parameter changes

2Productivity

If plasmid copy number is increased to improve gene expression levels, then productivity is improved, but metabolic burden increases

Engineering Contradiction:

Improvegene expression levelVSAvoidmetabolic burden

Core Design Contradiction:

ProductivityVSUse of energy by moving object

Solution Approach 1:

The modified inc RNA functions as a feedback regulator that responds to plasmid copy number changes. The specific nucleotide substitutions enhance the RNA's ability to regulate replication initiation, creating a feedback mechanism that maintains optimal copy numbers for high gene expression while preventing excessive replication that would increase metabolic burden.

Inventive Principle:

Principle #23Feedback

3Productivity

If plasmid replication is enhanced to achieve high target gene expression, then productivity is improved, but plasmid loss and mutation increase

Engineering Contradiction:

Improvetarget gene expressionVSAvoidplasmid segregation stability

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent introduces specific nucleotide changes in the inc RNA coding sequence that modify replication parameters to achieve high copy numbers while maintaining segregation stability. The C to U substitution at position 60 and other modifications tune the replication control mechanism to ensure faithful plasmid distribution during cell division, preventing plasmid loss and mutations even at high expression levels.

Inventive Principle:

Principle #35Parameter changes

Data Source

PatentUS11549116B2Nucleic acid molecules comprising a variant inc coding strand

Publication Date: 2023.01.10 CJ CHEILJEDANG CORP

AI summary

A nucleic acid molecule comprising a variant inc coding strand is disclosed as a regulator of plasmid copy number. Also disclosed is a replicon comprising the nucleic acid molecule, a promoter, and an origin of replication. Also disclosed is a vector comprising the replicon. Also disclosed is a recombinant microorganism comprising the vector.