A high-throughput platform for feedback-controlled directed evolution

Abstract

Continuous directed evolution rapidly implements cycles of mutagenesis, selection, and replication to accelerate protein engineering. However, individual experiments are typically cumbersome, reagent-intensive, and require manual readjustment, limiting the number of evolutionary trajectories that can be explored. We report the design and validation of Phage-and-Robotics-Assisted Near-Continuous Evolution (PRANCE), an automation platform for the continuous directed evolution of biomolecules that enables real-time activitydependent reporter and absorbance monitoring of up to 96 parallel evolution experiments. We use this platform to characterize the evolutionary stochasticity of T7 RNA polymerase evolution, conserve precious reagents with miniaturized evolution volumes during evolution of aminoacyl-tRNA synthetases, and perform a massively parallel evolution of diverse candidate quadruplet tRNAs. Finally, we implement a feedback control system that autonomously modifies the selection strength in response to real-time fitness measurements. By addressing many of the limitations of previous methods within a single platform, PRANCE simultaneously enables multiplexed, miniaturized, and feedback-controlled continuous directed evolution. from the National Cancer Institute (F32 CA247274-01).

Enabling high‐throughput biology with flexible open‐source automation

“…To pump media onto the deck, up to seven miniature 12 volt, 60 ml/min peristaltic pumps (“fish tank pumps”) were actuated by custom motor drivers. A Raspberry Pi mini single‐board computer received instructions over local IP and commanded the motor drivers via I 2 C (extended pump configuration details, see DeBenedictis et al, 2020). Between each iteration, the reservoir was filled with fresh 2XYT media, and then media was added to each bacterial turbidostat growing in a 24‐well plate, based on OD and parameter estimation.…”

Measuring the tolerance of the genetic code to altered codon size

“…Antibiotic selection markers have previously been used to identify functional qtRNAs 10 . We applied an equivalent approach based on the use of M13 bacteriophage tail fiber pIII as a selection marker 11 . For each of the 20 representative E. coli tRNA scaffolds used above, we created a 256-member qtRNA library containing degenerate anticodons (Figure 2A) and selected for functional qtRNAs from these libraries (Figure 2B) to identify qtRNAs that decode any of eight quadruplet codons of interest (Figure 2C).…”

“…Many qtRNAs were quite inefficient in translation: the presence of a single quadruplet codon in an mRNA transcript can reduce total protein yield to less than 3% relative to an all-triplet mRNA. Mutations at the anticodon loop sides of the qtRNAs have been observed to improve translation efficiency for TAGA-qtRNAs 11,13,36 . Triplet tRNAs are known to exhibit patterns that relate the bases in the ALS to the bases in the anticodon itself 14 , and similarly benefit from ALS mutations after anticodon replacement [15][16][17] .…”

Inducible Directed Evolution of Complex Phenotypes in Bacteria

“…Importantly, the use of temperate phages (such as P1) to passage variants to fresh hosts greatly reduces the impact of off-target mutations and decouples mutagenesis and screening steps, providing a large degree of flexibility when designing selections. In particular, we expect this approach to be highly amenable to automation, enabling rapid and highly parallel evolution campaigns similar to the eVOLVER 10 , PACE 18 , and PRANCE 20 systems. Taken together, IDE demonstrates that temperate phages are promising vehicles for directed evolution of complex phenotypes.…”

“…Although recent methods for directed evolution in bacteria have eliminated many hands-on steps via the use of filamentous phage (e.g. phageassisted continuous and non-continuous evolution (PACE, PANCE) [16][17][18] , phagemid-assisted continuous evolution (PACEmid) 19 and Phage-and-Robotics-Assisted Near-Continuous Evolution (PRANCE) 20 ), they are limited to small regions of DNA (<5kb) and often couple the mutagenesis and screening steps. This is because the phage used in these techniques (M13) has a strict packaging limit (5 kb) and is engineered to replicate as soon as a certain threshold of biological activity has been reached 21 .…”