Biology subjects

DeBenedictis, E. A.

Publications and source records attributed to DeBenedictis, E. A..

3 recordsLinked to original sources

bioRxiv2022

Randomized gates eliminate bias in sort-seq assays

Sort-seq assays are a staple of the biological engineering toolkit, allowing researchers to profile many groups of cells based on any characteristic that can be tied to fluorescence. However, current approaches, which segregate cells into bins deterministically based on their measured fluorescence, introduce systematic bias. We describe a surprising result: one can obtain unbiased estimates by incorporating randomness into sorting. We validate this approach in simulation and experimentally, and describe extensions for both estimating group level variances and for using multi-bin sorters.

bioengineering↗

bioRxiv2021

Measuring the tolerance of the genetic code to altered codon size

Protein translation using four-base codons occurs in both natural and synthetic systems. What constraints contributed to the universal adoption of a triplet-codon, rather than quadruplet-codon, genetic code? Here, we investigate the tolerance of the E. coli genetic code to tRNA mutations that increase codon size. We found that tRNAs from all twenty canonical isoacceptor classes can be converted to functional quadruplet tRNAs (qtRNAs), many of which selectively incorporate a single amino acid in response to a specified four-base codon. However, efficient quadruplet codon translation often requires multiple tRNA mutations, potentially constraining evolution. Moreover, while tRNAs were largely amenable to quadruplet conversion, only nine of the twenty aminoacyl tRNA synthetases tolerate quadruplet anticodons. These constitute a functional and mutually orthogonal set, but one that sharply limits the chemical alphabet available to a nascent all-quadruplet code. Our results illuminate factors that led to selection and maintenance of triplet codons in primordial Earth and provide a blueprint for synthetic biologists to deliberately engineer an all-quadruplet expanded genetic code.

synthetic biology↗

bioRxiv2020

A high-throughput platform for feedback-controlled directed evolution.

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.

bioengineering↗