Model-guided engineering of DNA sequences with predictable site-specific recombination rates

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

Site-specific recombination (SSR) is an important tool in synthetic biology, but its applications are limited by the inability to predictably tune SSR reaction rates. Facile rate manipulation could be achieved by modifying the DNA substrate sequence; however, this approach lacks rational design principles. Here, we develop an integrated experimental and computational method to engineer the DNA attachment sequence attP for predictably modulating the inversion reaction mediated by the recombinase Bxb1. After developing a qPCR method to measure SSR reaction rate, we design, select, and sequence attP libraries to inform a machine-learning model that computes Bxb1 inversion rate as a function of attP sequence. We use this model to predict reaction rates of attP variants in vitro and demonstrate their utility in gene circuit design in Escherichia coli. Our high-throughput, model-guided approach for rationally tuning SSR reaction rates enhances our understanding of recombinase function and expands the synthetic biology toolbox.

Original languageEnglish (US)
Article number4152
JournalNature communications
Volume13
Issue number1
DOIs
StatePublished - Dec 2022

Bibliographical note

Funding Information:
We thank Victor Garcia for assistance with next-generation sequencing, and Ayako Ohoka and Jennifer Kang for helpful discussions. This work was supported by the National Institutes of Health (R01DK114453 to S.M.A. and C.A.S. and R35GM136309 to C.A.S.).

Publisher Copyright:
© 2022, The Author(s).

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural

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