Crossland, P. | Search bioRxiv

bioRxiv2025

Massively parallel evolution reveals a biophysical scaling law between cell size and internal cell density

Using a massively parallel evolution platform, we selected Saccharomyces cerevisiae for increased cell size to test how cellular architecture adapts to biophysical constraints. As cells evolved larger size, they became less spherical and showed reduced carrying capacity without changes in maximum growth rate. Optical diffraction tomography revealed that individual cells with greater volume consistently exhibited lower internal density, a relationship that persisted across replicate populations and evolved isolates. The largest cells often contained enlarged vacuoles, suggesting that vacuole expansion may sometimes contribute to reduced density. Extending this analysis beyond yeast, 68 species spanning major phylogenetic clades also demonstrate a negative scaling between cell density and cell size. Together, these results suggest that decreased internal density is a conserved consequence of increasing cell size, revealing a fundamental cellular trade-off between volume expansion and material concentration.

biophysics↗

bioRxiv2025

INgen: Intracellular Genomic DNA Amplification for Downstream Applications in Sequencing and Sorting

Here, we introduce intracellular genomic amplification (INgen), a method that harnesses the cell membrane as a natural reaction chamber to amplify DNA within fixed, permeabilized cells. INgen employs a strand-displacing, isothermal polymerase together with biotinylated primers to achieve robust intracellular DNA amplification and efficient recovery of the amplified material for sequencing. This approach overcomes a long-standing barrier that has prevented important advances in single-cell and rare-cell genomics: the inability to amplify and recover sequenceable DNA from fixed cells. By pushing past this barrier, INgen provides a critical and previously inaccessible step toward scalable single-cell DNA sequencing without the need to isolate each cell into a separate reaction vessel. Using INgen, we demonstrate targeted and whole-genome amplification across diverse organisms, including Saccharomyces cerevisiae, Bacillus subtilis, and Escherichia coli. Together, these capabilities position INgen as a foundational advance that paves the way for the next generation of single cell sequencing methods including high-throughput single-cell sequencing without physical isolation, contamination-resistant amplification within intact cells, and rare-cell enrichment prior to genomic analysis.

molecular biology↗