Rapid Cell-Free Combinatorial Mutagenesis Workflow Using Small Oligos Suitable for High-Iteration, Active Learning-Guided Protein Engineering
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Abstract
Active learning-guided protein engineering e2iciently navigates the challenging fitness landscape by screening designs iteratively in a model-guided design-build-test-learn cycle. However, while high iterations boost performance, current workflows reliance on tedious and costly cell-based cloning and expression steps limits the iterations they can practically implement. To address this problem, we present a novel combinatorial mutagenesis workflow that uses small (∼20-40 bp) mutagenic annealed-oligo fragments and cell-free expression to rapidly and conveniently screen protein variants in <9 hours. Using bulk-prepared mutagenic oligos eliminates the need for cloning, PCR-based mutagenesis, or ordering costly genes each screening round. Their >80% size reduction from current fragment-based shu2ling strategies also helps avoid including multiple mutations on the same fragment, reducing the number one must order to cover the design space. By screening 3-10 fragment assemblies for two di2erent proteins, we show our approach is a general, scalable, and cost-e2ective platform for high-iteration protein engineering.
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Despite these limitations, our oligo-based cell-free screening workflow is well-suited to a wide-range of protein engineering tasks.
I really like this idea! Not necessarily in this paper, but I'm interested in seeing it stress-tested a bit more in future work. What happens with more complex oligos with more mutations? What happens in different proteins?
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This implies that even with misligation products, the assembly fidelity for the complex 10-oligo assembly is sufficient for cell-free protein screening.
Did you move these ligation products directly into cell-free testing like you did for the previous round, or did you do some kind of purification to eliminate those misligation products?
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< 9 hours using the low-cost Opentrons OT-2
How exciting!
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Specifically, we assayed a common batch size of 96 samples consisting of 6 replicates of 14 mutants, a missing oligo assembly, and a no DNA control.
Did you include a WT that you assembled similar to how you assembled the mutants?
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