Bacterial Chromosomal Engineering via Oligonucleotide and Plasmid Integration
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Solution Overview
Problem
Current genome engineering methods for bacteria, such as mycobacteria, are inefficient due to the laborious construction of long double-stranded DNA substrates and low recombination efficiency, while single-stranded oligonucleotides can make precise mutations but are not selectable and difficult to isolate.
Innovation Solution
A method using a combination of a targeting oligonucleotide with a Bxb1 phage integrase attP site and a non-replicating payload plasmid with an attB site, facilitated by single-strand annealing protein and integrase expression, for high-throughput chromosomal engineering that avoids the need for double-stranded DNA substrates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If double-stranded DNA substrates are used for recombination, then selectable mutations can be made, but the substrates are cumbersome to generate and recombine at low efficiency
Solution Approach 1:
The patent uses easily synthesized single-stranded oligonucleotides instead of cumbersome double-stranded DNA constructs. These short oligonucleotides are chemically synthesized, making them inexpensive and rapidly obtainable, while serving as temporary recombination substrates that are subsequently integrated into the genome
Solution Approach 2:
The patent changes the physical state of the recombination substrate from double-stranded DNA to single-stranded oligonucleotides. This parameter change enables easier synthesis and higher recombination efficiency while maintaining the ability to generate selectable mutations through the integration process
2Productivity
If single-stranded oligonucleotides are used for recombination, then precise mutations can be made at high efficiency, but the mutations are not selectable and difficult to isolate
Solution Approach 1:
The patent merges the advantages of single-stranded oligonucleotide recombination with selectable marker integration. The system combines the high efficiency of ssDNA recombination with the selectability of integrated markers by using a two-component system where the oligonucleotide provides precision and the integrated payload plasmid provides selection capability
3Adaptability or versatility
If long double-stranded DNA substrates with flanking homology are used, then diverse selectable mutations can be made, but the substrates are laborious to construct
Solution Approach 1:
The patent segments the recombination system into separate functional components: short homology-containing oligonucleotides for targeting and payload plasmids for mutation delivery. This segmentation eliminates the need to construct long dsDNA substrates for each mutation while maintaining versatility through modular design
Solution Approach 2:
The patent creates a universal system where a single payload plasmid backbone can be used with different oligonucleotide targets. The modular design allows the same basic system to generate diverse mutations across different genomic locations without requiring new substrate construction for each application
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables efficient and selectable genetic modifications in bacteria by integrating a selectable marker and payload into the chromosome, allowing for precise insertions and deletions with high efficiency and stability.
Implementation Method 1
a single strand annealing protein (SSAP) and/or an integrase protein is expressed in the bacterium
Implementation Method 2
a targeting oligo incorporates a Bxb1 phage integrase attP site at a target locus which allows simultaneous integration of a co-transfected payload plasmid comprising a cognate recombination site (e.g., attB site) and a selectable marker into the target locus
Data Source
AI summary
In some aspects, the disclosure relates methods and compositions for genetic engineering of cells (e.g., bacterial cells, etc.). The disclosure is based, in part, on a combination of genetic recombination systems (e.g., a “targeting oligonucleotide” and a “payload plasmid) that enable high-throughput chromosomal engineering and do not involve the preparation of double-stranded DNA (dsDNA) recombination substrates. In some aspects, the disclosure provides engineered bacterial cells comprising a targeting oligonucleotide and a payload plasmid.