Joshi, S. H.-N. | Search bioRxiv

bioRxiv2025

SHARK: a specialized host for assembling R6K plasmids

R6K plasmids are commonly used for a wide range of genome engineering applications due to their ability to support transient delivery of genetic cargoes in many hosts. The maintenance of R6K plasmids requires specific strains. Unfortunately, many of these have obscure backgrounds, limited availability and were not built for efficient cloning. To address this issue, we present the construction and characterisation of a series of Pir E. coli strains called SHARK that are built from the DH10B derivative, Marionette-Clo. All SHARK strains have a genome encoded Pir gene for stable R6K plasmid maintenance and a{lambda} CI gene for tight unconditional repression of specific genes on plasmids. We show that SHARK strains are >100-fold more efficient than a commercial Pir strain for large and complex cloning reactions. SHARK is intended to help facilitate the cloning of large and complex R6K plasmids for challenging genome engineering projects, with all strains and genetic tools for their assembly being made publicly available. TABLE OF CONTENTS IMAGE O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/680659v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@133c5e6org.highwire.dtl.DTLVardef@59532org.highwire.dtl.DTLVardef@8630b6org.highwire.dtl.DTLVardef@1e81abe_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Characterization of recombinase-based genetic parts and circuits using nanopore sequencing

Recombinases are versatile enzymes able to perform the precise insertion, deletion, and rearrangement of DNA and can act as a foundation for programmable genetic logic and memory. Fundamental to their use are accurate measurements of function. However, these are often laborious, time-consuming, and costly to collect. To address this, we developed a semi-automated workflow that combines low-cost liquid handling robotics, multiplexed long-read nanopore sequencing, and a supporting computational analysis tool to enable the high-throughput and detailed characterization of recombinase parts and circuits when used in a variety of contexts and organisms. Our approach overcomes the limitations of typically used fluorescence-based assays and is able to monitor temporal dynamics, observe structural changes at a nucleotide resolution, and unravel the internal workings of complex multi-state circuits. The ability to scale-up and automate genetic circuit characterization is an essential step towards more rigorous biological metrology that can support the construction of predictive models for efficiently engineering biology.

synthetic biology↗