Biology subjects

Mallon, J.

Publications and source records attributed to Mallon, J..

6 recordsLinked to original sources

bioRxiv2026

OpenEvo: An Open-Source Platform for Automated Evolution and Analysis

Here we introduce OpenEvo, a fully open-source, low-cost turbidostat platform for automated continuous culture and directed evolution experiments. Existing tools are expensive, complex, or lack open-source hardware; OpenEvo addresses this gap with a complete, fully automated evolution platform with detailed, illustrated construction instructions for beginners, open-source software and firmware, priced around $300. An optional PC-based interface offers enhanced functionality, including remote access, programmable evolution cycles, programmable LED stimulation, and a data visualization tool. OpenEvo can cycle through three types of media for positive, negative, and neutral selection conditions, supporting a wide range of experimental designs. We validate the use of OpenEvo by evolving Haloferax volcanii to grow from 15% to 12% salt over [~]150 cycles, [~]1,000 hours. Evolved cells grew 55% faster than wild-type at 12% salt. Whole-genome sequencing of adapted cells found SNPs and large deletions. We also demonstrate positive and negative selection using the OpenEvo LEDs to drive optogenetics via a Phytochrome B-based optogenetic tool, with light as the selection stimulus during over 4000 hours of growth. OpenEvo lowers the technical and cost barriers for continuous evolution experiments, serves as a teaching tool, and is designed to grow an open community of users who share modifications.

bioengineering↗

bioRxiv2024

Tissue-Like Multicellular Development Triggered by Mechanical Compression in Archaea

The advent of clonal multicellularity is a critical evolutionary milestone, seen often in eukaryotes, rarely in bacteria, and only once observed in archaea. We show that uniaxial compression induces clonal multicellularity in haloarchaea, forming tissue-like structures. These archaeal tissues are mechanically and molecularly distinct from their unicellular lifestyle, mimicking several eukaryotic features. Archaeal tissues undergo a multinucleate stage followed by tubulin-independent cellularization, orchestrated by active membrane tension at a critical cell size. After cellularization, tissue junction elasticity become akin to animal tissues and give rise to two cell types--radial peribasal and central apicobasal cells-- with distinct actin and protein glycosylation polarity patterns. Our findings highlight the potential convergent evolution of a biophysical mechanism in the emergence of multicellular systems across domains of life.

cell biology↗

bioRxiv2023

Salactin, a dynamically unstable actin homolog in Haloarchaea

Across the domains of life, actin homologs are integral components of many essential processes such as DNA segregation, cell division, and cell shape determination. Archaea genomes, like those of bacteria and eukaryotes, also encode actin homologs, but much less is known about these proteins in vivo dynamics and cellular functions. We identified and characterized the function and dynamics of Salactin, an actin homolog in the hypersaline archaeon Halobacterium salinarum. Despite Salactins homology to bacterial MreB proteins, we find it does not function as a MreB ortholog in H. salinarum. Rather, live-cell imaging revealed that Salactin forms dynamically unstable filaments that grow and shrink out of the cell poles. Like other dynamically unstable polymers, Salactin monomers add at the growing filament end and its ATP-bound critical concentration is substantially lower than the ADP-bound form. When H. salinarums chromosomal copy number becomes limiting under low phosphate growth conditions, cells lacking Salactin show perturbed DNA distributions. Taken together, we propose that Salactin is part of a previously unknown chromosomal segregation apparatus required during low-ploidy conditions.

microbiology↗

bioRxiv2019

Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

Cas9 has made a wide range of genome engineering applications possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity but at the cost of the on-target activity. DNA unwinding is the primary checkpoint before cleavage by Cas9 and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in Cas9. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two guanines added were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5 end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.

biophysics↗

bioRxiv2017

Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1

CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We have used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologues. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9, they both bind any DNA in search of PAM (protospacer-adjacent motif) sequences, verifies the target sequence directionally from the PAM-proximal end and rapidly rejects any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ~16 bp for cleavage, Cpf1 requires ~ 17 bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions and 5 guanine in guide-RNA differentially affected different Cpf1 orthologues. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

biophysics↗

bioRxiv2017

Mechanisms of improved specificity of engineered Cas9s revealed by single molecule analysis

In microbes, CRISPR-Cas systems provide adaptive immunity against invading genetic elements. Cas9 in complex with a guide-RNA targets complementary DNA for cleavage and has been repurposed for wide-ranging biological applications. New Cas9s have been engineered (eCas9 and Cas9-HF1) to improve specificity, but how they help reduce off-target cleavage is not known. Here, we developed single molecule DNA unwinding assay to show that sequence mismatches affect cleavage reactions through rebalancing the internal unwinding/rewinding equilibrium. Increasing PAM-distal mismatches facilitate rewinding, and the associated cleavage impairment shows that cleavage proceeds from the unwound state. Engineered Cas9s depopulate the unwound state more readily upon mismatch detection. Intrinsic cleavage rate is much lower for engineered Cas9s, preventing cleavage from transiently unwound off-targets. DNA interrogation experiments showed that engineered Cas9s require about one additional base pair match for stable binding, freeing them from sites that would otherwise sequester them. Therefore, engineered Cas9s achieve their improved specificity (1) by inhibiting stable DNA binding to partially matching sequences, (2) by making DNA unwinding more sensitive to mismatches, and (3) by slowing down intrinsic cleavage reaction.

biophysics↗