Abstract
Engineering regulatory parts for improved performance in genetic programs has played a pivotal role in the development of the synthetic biology cell programming toolbox. Here, we report the development of a novel high-Throughput platform for regulatory part prototyping and analysis that leverages the advantages of engineered DNA libraries, cell-free protein synthesis (CFPS), high-Throughput emulsion droplet microfluidics, standard flow sorting adapted to screen droplet reactions, and next-generation sequencing (NGS). With this integrated platform, we screened the activity of millions of genetic parts within hours, followed by NGS retrieval of the improved designs. This in vitro platform is particularly valuable for engineering regulatory parts of nonmodel organisms, where in vivo high-Throughput screening methods are not readily available. The platform can be extended to multipart screening of complete genetic programs to optimize yield and stability.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 2108-2120 |
| Number of pages | 13 |
| Journal | ACS synthetic biology |
| Volume | 11 |
| Issue number | 6 |
| DOIs | |
| State | Published - Jun 17 2022 |
Funding
This work was funded by the U.S. Defense Advanced Research Projects Agency’s (DARPA) Living Foundries program Award HR0011-15-C-0084. M.C.J. also gratefully acknowledges the Packard Foundation.
Keywords
- DNA library
- T7 promoter
- cell-free protein synthesis
- emulsion droplet
- flow cytometry
- microfluidics
- next-generation sequencing
- regulatory parts
- ribosome binding site
ASJC Scopus subject areas
- Biomedical Engineering
- Biochemistry, Genetics and Molecular Biology (miscellaneous)