Plasmid Vectors for Stable In Vivo Protein Secretion
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Solution Overview
Problem
Current methods for engineering probiotic bacteria like E. coli Nissle for therapeutic use in the gut face challenges such as plasmid loss without antibiotic selection, inefficient genomic incorporation, and limitations in achieving high gene expression due to poor transformation efficiencies and insert length limitations, hindering the development of stable and effective synthetic genetic systems.
Innovation Solution
Engineering an E. coli strain with modified cryptic plasmids that remain stably associated without antibiotic selection, enabling protein secretion in the gastrointestinal tract, and utilizing a temperature-sensitive gene expression system for efficient recombinant protein production and secretion.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If plasmid vectors are used for genetic engineering of E. coli Nissle, then transformation efficiency and gene expression levels are improved, but plasmid stability is worsened due to loss without antibiotic selection
Solution Approach 1:
The patent changes the physical state of the plasmid by integrating it into the bacterial chromosome, transforming it from an extrachromosomal element to a genomic element. This integration ensures stable inheritance without antibiotic selection while maintaining the ability to achieve high gene expression through multiple copy integration or strong promoter usage.
Solution Approach 2:
The patent merges the plasmid DNA with the bacterial chromosome through integration events. This combining of previously separate genetic elements (plasmid and chromosome) creates a stable, inheritable construct that eliminates plasmid loss while preserving the benefits of plasmid-based gene expression.
2Stability of the object's composition
If genomic incorporation is used to maintain plasmid stability, then plasmid loss is prevented, but transformation efficiency and gene expression levels are worsened
Solution Approach 1:
The patent allows for partial integration where multiple plasmid copies can integrate into the chromosome, or the entire plasmid integrates as a unit. This partial or complete integration approach maintains stability while the multiple copy number or strong promoters can compensate to achieve high gene expression levels, thus not fully sacrificing productivity.
3Stability of the object's composition
If antibiotic selection is used to maintain plasmids, then plasmid stability is improved, but in vivo therapeutic compatibility is worsened due to immunosuppression and microbiome disruption
Solution Approach 1:
The patent extracts and eliminates the antibiotic selection marker from the plasmid construct. By removing this harmful element, the system achieves plasmid stability through chromosomal integration without requiring antibiotic pressure, thus eliminating the harmful effects on the host microbiome and immune system while maintaining the therapeutic function.
4Stability of the object's composition
If chromosomal integration is used for genetic engineering, then stable transformants are achieved, but transformation efficiency and engineering speed are worsened
Solution Approach 1:
The patent utilizes the bacterium's own natural competence and integration mechanisms to achieve chromosomal integration. By leveraging endogenous cellular processes rather than requiring complex external integration systems, the method achieves stable integration with improved transformation efficiency and faster engineering cycles.
Data Source
AI summary
Disclosed are methods for producing genetically modified bacteria, comprising introducing into said bacteria at least one engineered cryptic plasmid comprising a heterologous nucleic acid, wherein the heterologous nucleic acid comprises a nucleic acid sequence encoding a recombinant protein and a polypeptide secretion system for directing the recombinant protein to the outer membrane for secretion, wherein the bacteria do not comprise any native cryptic plasmids.