AI Article Synopsis
- Efficient phage production is essential for drug discovery and gene evolution, prompting the development of a faster cell-free synthesis system for M13 phage by simplifying its genome.* -
- A new cell-free directed evolution system using a modified helper plasmid and a gene mutation library was created, which works more effectively with fluorescence-activated droplet sorting (FADS).* -
- This system successfully evolved T7 RNA polymerase to double its activity and enhanced tryptophan tRNA into a suppressor tRNA with an eightfold increase in activity.*
Article Abstract
Efficient phage production has always been an urgent need in fields such as drug discovery, disease treatment, and gene evolution. To meet this demand, we constructed a robust cell-free synthesis system for generating M13 phage by simplifying its genome, enabling a three-times faster efficiency compared with the traditional method in vivo. We further developed a cell-free directed evolution system in droplets, comprising a modified helper plasmid (ΔPS-ΔgIII-ΔgVI) and the simplified M13 genome-carrying gene mutation library. This system was greatly improved when coupled with fluorescence-activated droplet sorting (FADS). We successfully evolved the T7 RNA polymerase (RNAP), achieving a twofold higher activity to read through the T7 terminator. Moreover, we evolved the tryptophan tRNA into a suppressor tRNA with an eightfold increase in activity to read through the stop codon UAG.
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| http://dx.doi.org/10.1016/j.tibtech.2024.10.005 | DOI Listing |
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