Abstract
Cell-free synthetic biology enables rapid prototyping of biological parts and synthesis of proteins or metabolites in the absence of cell growth constraints. Cell-free systems are frequently made from crude cell extracts, where composition and activity can vary significantly based on source strain, preparation and processing, reagents, and other considerations. This variability can cause extracts to be treated as black boxes for which empirical observations guide practical laboratory practices, including a hesitance to use dated or previously thawed extracts. To better understand the robustness of cell extracts over time, we assessed the activity of cell-free metabolism during storage. As a model, we studied conversion of glucose to 2,3-butanediol. We found that cell extracts from Escherichia coli and Saccharomyces cerevisiae subjected to an 18-month storage period and repeated freeze-thaw cycles retain consistent metabolic activity. This work gives users of cell-free systems a better understanding of the impacts of storage on extract behavior.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 904-908 |
| Number of pages | 5 |
| Journal | ACS synthetic biology |
| Volume | 12 |
| Issue number | 3 |
| DOIs | |
| State | Published - Mar 17 2023 |
Funding
This work was funded by an Emerging Technologies Opportunity Program (ETOP) award under Subcontract No. 7399340 from the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility supported under Contract No. DE-AC02-05CH11231. B.J.R. was supported by a National Defense Science and Engineering Graduate Fellowship (Award ND-CEN-017-095).
Keywords
- biocatalysis
- cell-free
- extract
- in vitro
- metabolism
- stability
ASJC Scopus subject areas
- Biomedical Engineering
- Biochemistry, Genetics and Molecular Biology (miscellaneous)
