One of the most popular applications of synthetic biology is to confer desired traits to an organism. Designing a range of possibilities and then testing each one for fitness, productivity, stability, or some other goal is critically important as the use of synthetic biology becomes more mainstream.
Many scientists accomplish this with targeted libraries for directed evolution of enzymes, so we thought it would make a great webinar topic. Katie Lyons, a senior scientist at Codex DNA, teamed up with Russell Komor, Director of Biochemistry at Cellibre, to offer a helpful view of directed evolution and targeted libraries.
If you missed the live webinar, you can watch the on-demand recording or check out the highlights we’ve recapped here.
What’s a Codex DNA library?
Lyons began with noting that people have different understandings of what a DNA library is, so she set the stage with our definition — a series of DNA fragments with variations in one or more positions that share at least 85% sequence homology.
To determine what type of DNA library is best for your project, Lyons said it’s important to determine scale and format. Lower-capacity needs can be easily run on a lab’s BioXp™ system, while larger-scale projects might be more feasible when ordered directly from Codex DNA variant library services. The format consideration boils down to grouped or targeted libraries. Each well contains one library of sequences in the former, while in the latter, each well contains one specific sequence.
Targeted libraries are one type of DNA library offered by Codex DNA variant library services that include specific mutations distributed over the sequence space to achieve the desired diversity. Targeted libraries are synthesized with patented error-correction technology, so libraries have the lowest error rate in the industry — less than 1:10,000 base pairs. Target mutations can be studied singly or in parallel, with no restrictions on positioning. This makes targeted libraries well suited for enzyme engineering.
At Cellibre, scientists are using synthetic biology to make products more sustainably and efficiently. They have a major focus on cannabinoids, which represent a market valued at more than $500 billion, but are currently limited by inefficient, expensive, and low-quality production methods. By optimizing pathways such as CBGA production, Komor and his colleagues hope to find a better way to make CBD, THC, and potentially other cannabinoids in advantaged host organisms. The enzyme engineering process they use begins with enzyme discovery, through which a template is identified for iterative cycles of engineering to ultimately produce a candidate enzyme. Each engineering cycle involves library design, screening, and data analysis. These engineering cycles are sequential, as the output of the previous cycle serves as input for the next cycle. This makes template selection a rate-limiting step. Strategies to compress the time taken for each iterative cycle are key to minimizing the time taken to identify the top candidates.
Better library design
Library construction can be time-consuming. Turnaround time for library services with certain providers is as long as six weeks. Komor’s team needed a way to speed up the library construction step, which typically includes design, synthesis, cloning, transformation, culturing, sequencing, and assaying. Reducing the time allocated to these steps would make it possible to test more variants for faster design-build-test cycles.
Komor worked closely with the Codex DNA team to design and synthesize targeted libraries, site-saturation libraries, as well as fragments, to obtain high fidelity expression constructs needed for their enzyme engineering. With turnaround times as low as a week, Komor and team were able to significantly shorten design-build-test cycles and identify top enzyme candidates with dramatically improved CBGA production. The use of error-correction technology to synthesize targeted libraries resulted in additional time and resource savings by allowing his team to omit steps required for clonality and sequence verification.
The most impressive improvement, he said, came from adding multiple mutations in different regions of the enzyme — a discovery enabled by the Codex DNA approach.
“Targeted libraries are a very powerful product — you can do quite a lot,” Komor told webinar attendees. “I’ve actually contacted other companies with the exact same design… and they don’t even know where to start.”