We will employ standard genetic engineering methods to move the open reading frames of the enzymes into expression vectors. These methods may include classical restriction enzyme digestion/ligations, NEbuilder and Golden Gate assembly. Open Bioeconomy has provided starter protocols in an Open Enzyme Expression Guide for all the enzymes in the FreeGenes Open Enzymes collections, including a suggested expression vector and purification methods. It will be our job to determine the cheapest and most efficient methods for a community lab setting. In order to become an efficient reagent hub, we will research best practices from other organizations that distribute tools and create replicable models that facilitate capacity in a distributed manner.
The most difficult part of the project will be the expression optimization. We anticipate continuing to improve on expression methods past the end of this project since we anticipate that not all of the enzymes in the collection will be easy or cost-effective to produce and purify. Certain components of standard purification methods may be difficult to obtain in low-resource labs. We intend to keep abreast of any new developments and explore multiple methods beyond classic affinity columns (bead-based purification, cell-free systems etc.).
This study is non-quantitative at the top level. We are documenting our process to become a distribution node and sharing our learnings. We may be only able to produce a portion of the enzymes in the collection within this six month period, although our goal is to try them all in parallel. At minimum we should be able to move all the Open Enzymes collection into expression vectors and distribute these materials. We will compare different workflows for production and distribution within a community lab setting. Success will be determined based on the percentage of enzymes we can successfully 1) move into expression vectors 2) express such that they function and 3) distribute to at least one other lab or classroom. Publishing the guide on our website and the number of views within the next year will also confirm success of the project.
This project has not yet shared any protocols.