Plasmid Copy Number Enhancement via SV40 Enhancer Integration
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Solution Overview
Problem
Current methods for producing covalently closed circular recombinant DNA molecules, such as plasmids, face challenges in achieving high yields during fermentation, which affects product purity and increases production costs.
Innovation Solution
Modifying plasmid DNA by incorporating an SV40 enhancer, PAS-BH region, or removing transcriptional terminators, and transforming these modified plasmids into bacterial cells to enhance plasmid copy number and yield in shake flask and fermentation cultures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional plasmid production methods are used, then production costs are incurred, but plasmid yield and purity are insufficient
Solution Approach 1:
The patent modifies plasmid DNA sequences by changing specific parameters such as adding SV40 enhancer elements, modifying promoter regions, and adjusting origin of replication sequences to optimize copy number and expression levels, thereby simultaneously improving yield and purity
Solution Approach 2:
The invention creates composite plasmid structures by combining multiple functional elements including SV40 enhancer sequences, bacterial origin of replication, promoter regions, and gene of interest, forming a hybrid construct that achieves both high yield and high purity through synergistic interactions among components
2Productivity
If plasmid copy number is increased, then production yield improves, but production cost increases
Solution Approach 1:
The modified plasmids contain self-regulating elements such as optimized origin of replication sequences and copy number control regions that automatically maintain optimal copy numbers during bacterial growth, eliminating the need for external intervention or additional processing steps to achieve high yields
Solution Approach 2:
The plasmid DNA is pre-modified with optimized sequences including SV40 enhancers and improved origin regions before transformation, so that the bacterial cells inherently produce high yields of plasmid DNA without requiring costly post-culture optimization or additional production enhancements
3Quantity of substance
If fermentation conditions are optimized, then plasmid purity improves, but production yield may be affected
Solution Approach 1:
The patent applies local quality optimization by specifically enhancing the plasmid backbone sequence with SV40 enhancer elements and optimized origin regions at critical locations, while leaving the gene of interest and other functional regions unchanged, thereby improving overall yield and purity without affecting fermentation conditions
Data Source
AI summary
The present invention relates to the production of covalently closed circular (ccc) recombinant DNA molecules such as plasmids, cosmids, bacterial artificial chromosomes (BACs), bacteriophages, viral vectors and hybrids thereof, and more particularly to vector modifications that improve production yield of said DNA molecules in fermentation culture.