Genomically Recoded Cells for Stable Toxic Protein Production
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Solution Overview
Problem
Existing methods for maintaining and manipulating DNA sequences in microbial host cells, such as E. coli, face challenges due to toxicity and metabolic burden from DNA sequences, particularly eukaryotic or viral sequences, which are unstable and difficult to clone due to cryptic bacterial promoters, leading to expression of toxic proteins or peptides.
Innovation Solution
Genomically recoded cells or organisms are created by altering native genes to eliminate tRNA cognates for specific codons, rendering them unassigned, and introducing foreign nucleic acid sequences containing these codons, which prevents initial translation and allows for controlled expression through exogenous tRNA supply, enabling stable propagation and high-yield protein production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If foreign nucleic acid sequences (eukaryotic or viral) are introduced into microbial host cells, then the ability to produce diverse proteins is improved, but stability and cloning ease deteriorate due to cryptic bacterial promoters causing toxic protein expression
Solution Approach 1:
The patent extracts and removes cryptic bacterial promoters from foreign nucleic acid sequences through site-directed mutagenesis or promoterless vector design, eliminating the source of toxic protein expression while preserving the coding sequences for desired protein production
Solution Approach 2:
The patent introduces intermediary regulatory elements such as inducible promoters (e.g., T7 promoter with lac operator) that act as mediators between the foreign nucleic acid sequences and the host cell's transcriptional machinery, allowing controlled expression only when needed
2Ease of manufacture
If cryptic bacterial promoters in foreign sequences are left intact, then cloning simplicity is improved, but metabolic burden increases due to expression of toxic proteins or peptides
Solution Approach 1:
The patent converts the potentially harmful effect of cryptic promoters into a beneficial selection mechanism by designing systems where promoter activity serves as a marker for successful cloning events, allowing easy identification of correctly transformed cells while controlling toxic expression
3Productivity
If DNA sequences are maintained in microbial host cells, then productivity for protein production is improved, but metabolic burden increases leading to reduced cell growth and stability
Solution Approach 1:
The patent implements periodic or inducible expression systems where foreign genes are expressed only at specific times or under specific conditions (e.g., addition of IPTG, temperature shift), allowing cells to grow normally during propagation and produce proteins only when induced, thereby reducing overall metabolic burden
Data Source
AI summary
Methods making and using genomically recoded cells or organisms are provided including genomically recoded cells or organisms that lack the ability to translate a foreign nucleic acid sequence into a polypeptide that may be toxic to the genomically recoded cell or organism.