Gutierrez-Lopez, L. | bioRxiv Science

bioRxiv2025

Essentialome-Wide Multigenerational Imaging Reveals Mechanistic Origins of Cell Growth Laws

Escherichia coli is arguably the most thoroughly characterized organism, and yet [~]20% of its essential genes serve completely unknown functions,1,2 and many core quantitative physiological principles remain unexplained, including the famous nutrient growth law where cell volume seems to depend exponentially on growth rate. Here we develop a platform for massive, multi-generational Optical Pooled Screening (OPS)3-6, and apply it to link image-based phenotypes to genotypes for 133,000 CRISPRi knockdowns of essential genes, tracking tens of millions of lineages and analyzing 1.6 billion cells. Our multi-dimensional dynamic phenotypes correlate exceptionally well with known gene functions, allowing us to identify many unknown roles of essential genes. Quantifying the relation between growth and cell size in turn identifies three distinct variants of the bacterial growth laws, which we explain mechanistically by discovering a new role for (p)ppGpp and SpoT as a sensor of translation elongation. Finally, we propose and systematically test an exceedingly simple, passive mechanism for the nutrient growth law, based on triggering cell division through the accumulation of a protein that, unlike ribosomes, is not controlled by (p)ppGpp. The resulting hyperbolic growth law fits the data from our three variants even better than the previously proposed exponential relationship, and with fewer parameters.

systems biology↗

bioRxiv2023

BatCRISPRi: Bacillus titratable CRISPRi for dynamic control in Bacillus subtilis

The discovery of new genes regulating essential biological processes has become increasingly important, and CRISPRi has emerged as a powerful tool for achieving this goal. This method has been used in many model organisms to decrease the expression of specific genes and assess their impact on phenotype. Pooled CRISPRi libraries in bacteria have been particularly useful in discovering new regulators of growth, division, and other biological processes. However, these libraries rely on the induction of dCas9 via an inducible promoter, which can be problematic due to promoter leakiness. This is a widespread phenomenon of any inducible promoter that can result in the unwanted downregulation of genes and the emergence of genetic suppressors when essential genes are knocked down. To overcome this issue, we have developed a novel strategy that eliminates dCas9 leakiness and enables reversible knockdown control using the rapamycin-dependent degron system in Bacillus subtilis. This degron system causes rapid degradation of dCas9, resulting in an almost instant reset of the system. Our results demonstrate that it is possible to achieve zero CRISPRi activity in the uninduced state and full activity in the induced state. This improved CRISPRi system will enable researchers to investigate phenotypic changes more effectively while reducing the undesirable effects of leaky expression and noise in their phenotypic data. Moreover, a rapid degradation system could serve as a tool for dynamic perturbation before compensation mechanisms or stress responses kick in. Finally, this approach can be adapted to other organisms and other promoter-inducible systems, potentially opening up strategies for tighter control of gene expression.

synthetic biology↗