Synthetic Genome Codon Compression for Viable Prokaryotic Cells
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Solution Overview
Problem
Existing methods for genome-wide synonymous codon compression in prokaryotic genomes have not been able to produce viable organisms with a significantly reduced number of sense codons, and there is a need for improved methods to produce synthetic genomes with minimal sense codons.
Innovation Solution
A method combining recombination-mediated genetic engineering (REXER and GENESIS) with directed conjugation is used to efficiently replace large portions of a prokaryotic genome, allowing for genome-wide synonymous codon compression, reducing sense codons to 5 or fewer occurrences, and replacing stop codons to achieve a viable synthetic prokaryotic genome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If site-directed mutagenesis is used to replace target codons, then up to 321 amber stop codons can be replaced, but sense codons which are orders of magnitude more abundant cannot be effectively tackled
Solution Approach 1:
The genome is divided into multiple synthetic DNA fragments that are synthesized separately and then assembled together. This allows systematic replacement of abundant sense codons throughout the genome by dividing the large-scale replacement task into manageable fragment-level operations, making genome-wide codon compression feasible
Solution Approach 2:
The patent replaces traditional site-directed mutagenesis methods with genome synthesis approaches. Instead of using enzymatic mutagenesis for each codon replacement, the entire genome or large portions are synthesized de novo with the desired codon substitutions already incorporated, dramatically increasing the scale and efficiency of codon replacement
2Quantity of substance
If previous recoding schemes are applied to small fractions of the genome, then up to 4.7% of targeted sense codons can be mutated, but it remains unknown whether these methods can produce viable genomes with genome-wide codon compression
Solution Approach 1:
The patent performs preliminary testing and optimization of recoding schemes on smaller genomic regions before applying them genome-wide. Synthetic DNA fragments with compressed codons are first tested for functionality and organism viability, allowing validation of recoding schemes before full-scale genome replacement, thus ensuring reliability of the final compressed genome
Solution Approach 2:
The patent incorporates feedback mechanisms where the effects of codon compression on organism viability are monitored and used to guide further genome synthesis and recoding. Data from partial genome replacements inform adjustments to recoding schemes, ensuring that genome-wide compression maintains organism functionality
3Length of stationary object
If genome synthesis is used to replace large portions of DNA, then over 1 Mb can be replaced in individual strains, but the complexity and difficulty of producing viable synthetic genomes with reduced sense codons increases
Solution Approach 1:
The large-scale genome replacement is achieved by dividing the genome into multiple synthetic DNA fragments of manageable size. Each fragment is synthesized separately with the desired codon compressions, then assembled into the complete genome through standardized assembly procedures, making the complex task of replacing over 1 Mb of DNA more tractable
Solution Approach 2:
The patent develops universal methods and standardized protocols for synthetic DNA fragment assembly that can be applied regardless of the specific organism or target codons. This modular, universal approach reduces the complexity of genome synthesis by creating reusable techniques that work across different genomic contexts
Data Source
AI summary
The current invention provides a synthetic prokaryotic genome comprising 5 or fewer occurrences of one or more sense codons; and/or a synthetic prokaryotic genome derived from a parent genome, wherein the synthetic prokaryotic genome comprises less than 10%, 5%, 2%, 1%, 0.5%, 0.1% of the occurrences of one or more sense codons, relative to the parent genome; and/or a synthetic prokaryotic genome comprising 100 or more, 200 or more, or 1000 or more genes with no occurrences of one or more sense codons.