Directed evolution of Escherichia coli with lower-than-natural plasmid mutation rates
| Autor: | Jeffrey E. Barrick, Daniel E. Deatherage, Álvaro E. Rodriguez, Dacia Leon, Salma K. Omar |
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| Rok vydání: | 2018 |
| Předmět: |
DNA Replication
DNA Bacterial 0301 basic medicine Mutation rate DNA Copy Number Variations Ultraviolet Rays Biology medicine.disease_cause Genome engineering 03 medical and health sciences Synthetic biology Plasmid Mutation Rate Genome editing Endoribonucleases Escherichia coli Genetics medicine Selection Genetic 030304 developmental biology 2. Zero hunger Mutation 0303 health sciences ColE1 Base Sequence 030102 biochemistry & molecular biology Escherichia coli Proteins 030302 biochemistry & molecular biology Sequence Analysis DNA DNA Polymerase I Directed evolution 030104 developmental biology biology.protein Synthetic Biology Directed Molecular Evolution DNA polymerase I Genetic Engineering Synthetic Biology and Bioengineering Plasmids |
| Zdroj: | Nucleic Acids Research |
| Popis: | Unwanted evolution of designed DNA sequences limits metabolic and genome engineering efforts. Engineered functions that are burdensome to host cells and slow their replication are rapidly inactivated by mutations, and unplanned mutations with unpredictable effects often accumulate alongside designed changes in large-scale genome editing projects. We developed a directed evolution strategy, Periodic Reselection for Evolutionarily Reliable Variants (PResERV), to discover mutations that prolong the function of a burdensome DNA sequence in an engineered organism. Here, we used PResERV to isolate E. coli cells that replicate ColE1 plasmids with higher fidelity. We found mutations in DNA polymerases I and IV and in RNase E that reduce plasmid mutation rates by 6-to 30-fold. The PResERV method implicitly selects to maintain the growth rate of host cells, and high plasmid copy numbers and gene expression levels are maintained in some of the evolved E. coli strains, indicating that it is possible to improve the genetic stability of cellular chassis without encountering trade-offs in other desirable performance characteristics. Utilizing these new antimutator E. coli and applying PResERV to other organisms in the future promises to prevent evolutionary failures and unpredictability to provide a more stable genetic foundation for synthetic biology. |
| Databáze: | OpenAIRE |
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