Codon-Optimized PheS Counterselection for Scarless Bacillus Editing
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Solution Overview
Problem
Current methods for genetic engineering in Bacillus species face challenges due to the ineffectiveness of existing counterselectable markers, leading to low loop-out rates and difficulties in achieving scarless genomic edits, especially in high-throughput contexts, as they often require pre-existing genetic mutations or result in false-positive clones.
Innovation Solution
The development of a high-throughput method using a plasmid or linear DNA construct with a counterselectable marker based on the α-subunit of Phenylalanyl-tRNA ligase (PheS) optimized for Bacillus, featuring A309G/T255S mutations, which is operably linked to a promoter and used in conjunction with positive selection to facilitate scarless genomic edits by promoting homologous recombination and excision of the marker backbone.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing counterselectable markers (such as PheS with A294G/T251A mutations) are used in Bacillus species, then the marker can be used for counterselection, but the counterselection efficiency is low and false-positive clones occur
Solution Approach 1:
The patent applies parameter changes by modifying the PheS gene sequence through specific mutations (A309G and T255S in Bacillus, corresponding to A294G and T251A in E. coli) and codon optimization to adapt the counterselectable marker to Bacillus species. These parameter changes in the gene sequence improve counterselection efficiency while reducing false-positive clones, resolving the contradiction between reliability and measurement precision.
2Reliability
If pre-existing genetic mutations (such as upp or pyrF deletions) are required for counterselection, then counterselection can be achieved, but the method complexity and preparation time increase
Solution Approach 1:
The patent extracts the counterselection functionality from the requirement of pre-existing genetic mutations by providing a standalone counterselectable marker (PheS with specific mutations) that can be introduced via plasmid or linear DNA construct without needing upp or pyrF deletions. This takes out the complexity of preparing strains with pre-existing mutations while maintaining reliable counterselection functionality.
3Manufacturing precision
If loop-out rates are low (0.8% to 5% without effective counterselection), then genomic edits can be constructed, but the productivity and efficiency of high-throughput genetic engineering decrease
Solution Approach 1:
The patent implements feedback through the counterselectable marker system that provides real-time selection pressure during the genetic engineering process. The PheS marker with A309G/T255S mutations enables efficient counterselection that feeds back to eliminate unwanted clones (those that did not undergo loop-out), thereby increasing both the accuracy of genomic edits and the productivity of high-throughput experiments by enriching for successful loop-out events.
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
This approach significantly increases the efficiency of scarless genomic editing in Bacillus species, achieving high loop-out rates and minimizing false positives, thereby enhancing the precision and predictability of genetic modifications.
Implementation Method 1
selecting for a microorganism having undergone a homologous recombination event excising the backbone of the plasmid containing the counterselectable markers
Data Source
AI summary
The present disclosure is directed to methods of scarless genomic engineering in microorganisms, such as Bacillus, and provides for new molecular tools and methods which enable scarless genetic editing using at least one counterselectable marker that has been codon optimized for the microorganism. The disclosure allows for the high-throughput introduction of stable genetic edits to a genome using either plasmid or linear DNA constructs for genetic engineering.