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Open Access Publications from the University of California

Scholarly Works (21 results)

  • Article
  • Peer Reviewed
UC San Diego Previously Published Works (2015)
    Cover page: Chromosome, Cell Cycle, and Entropy
    • Thesis
    • Peer Reviewed
    UC San Diego Electronic Theses and Dissertations (2018)

    The defining feature of living organisms is their capacity to reproduce and pass on the genetic information so that their progeny can flourish. For bacteria, reproduction is a feat by itself – Escherichia coli cells cultured in optimal conditions grow rapidly and divide about every 20 minutes. In other words, the cell has to replicate all cellular contents, and be ready to divide evenly into two daughter cells within this 20 minutes. Biosynthesis of new cellular materials, e.g. proteins, nucleic acis, lipids and other metabolites accumulate and roughly doubles after every generation. Notably, the deoxyribonucleic acid (DNA) encodes genetic informationand needs to be duplicated in order to faithfully pass on this information to the progeny. This process of DNA replication in the cell needs to dynamically adapt to fluctuation in growth condition and cellular physiology. Such coordination is controlled at the first step of replcation – the initiation of replication. In this thesis, I presented the development of methods for measuring DNA replication duration (replication period), the quantitative relationship between DNA replication and cell size as well as the mechanism of replication initiation. DNA replication measurement laid the foundation of studying the quantitative relationship between cell size and DNA replication. A general growth law was proposed to describe cell size regulation in light of three physiological variables including biosynthesis rate, cell cycle progression and replicaiton initiation. Of the three variables, the mass when cell initiates replication (initiation mass) remains invariant despite a wide spectrum of antibiotics or growth limitation challenge. This invariant initiation mass called into question about the mechanism of initiation to achieve such constancy. We proposed a simple threshold model to explain how cells can maintain a invariant initiation mass by regulating the expression of initiation regulators (initiators). The initiaiton mass is inversely proportional to the initiator levels, which is held constant. Experimental evidence was provided to test our model prediction.

      Cover page: DNA replication and cell size control in Escherichia coli
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2015)
      Escherichia coli NCM3722 is a prototrophic K-12 strain with robust physiologic phenotypes. We report the complete 4,678,045-bp chromosome and 67,545-bp F-like plasmid of this unique model organism.
        Cover page: Complete Genome Sequence of Escherichia coli NCM3722
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2015)
        How cells maintain a stable size has fascinated scientists since the beginning of modern biology, but has remained largely mysterious. Recently, however, the ability to analyze single bacteria in real time has provided new, important quantitative insights into this long-standing question in cell biology.
          Cover page: Cell-size maintenance: universal strategy revealed
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2017)
          We present a protocol for construction of tunable CRISPR interference (tCRISPRi) strains for Escherichia coli. The tCRISPRi system alleviates most of the known problems of plasmid-based expression methods, and can be immediately used to construct libraries of sgRNAs that can complement the Keio collection by targeting both essential and nonessential genes. Most importantly from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step oligo recombineering. Additional advantages of tCRISPRi over other existing CRISPRi methods include: (1) tCRISPRi shows significantly less than 10% leaky repression; (2) tCRISPRi uses a tunable arabinose operon promoter and modifications in transporter genes to allow a wide dynamic range with graded control by arabinose inducer; (3) tCRISPRi is plasmid free and the entire system is integrated into the chromosome; (4) tCRISPRi strains show desirable physiological properties.
            Cover page: Protocol for Construction of a Tunable CRISPR Interference (tCRISPRi) Strain for Escherichia coli.
            • Thesis
            • Peer Reviewed
            UC San Diego Electronic Theses and Dissertations (2019)

            Each cell must achieve the dual tasks of replicating the genetic material and dividing its components into its daughters. This thesis is concerned with how the cell coordinates those fundamental requirements. It approaches this question from the perspective of bacterial physiology, looking at the global relationship between these processes and growth at large. The thesis aims to uncover and test quantitative principles which govern this physiological orchestration. We first review the early quantitative principles, or growth laws, and their modern expansions. This includes cell size control, its connection with the cell cycle, and homeostatic control of each. We then explore these principles in the context of the model organisms E. coli and B. subtilis. Despite their evolutionary distance, we find the two species to be fundamentally similar in their physiological control. Finally, we test the limits of replication and division coordination using single-cell measurements and nutrient-shift experiments. This leads us to reevaluate the canonical Helmstetter-Cooper cell cycle model. Instead of a strict dependency of division on replication initiation, we instead support the view that both processes are controlled by independent threshold mechanisms.

              Cover page: Physiological Orchestration in Bacteria
              • Article
              • Peer Reviewed
              UC San Diego Previously Published Works (2023)
              Balanced biosynthesis is the hallmark of bacterial cell physiology, where the concentrations of stable proteins remain steady. However, this poses a conceptual challenge to modeling the cell-cycle and cell-size controls in bacteria, as prevailing concentration-based eukaryote models are not directly applicable. In this study, we revisit and significantly extend the initiator-titration model, proposed 30 years ago, and we explain how bacteria precisely and robustly control replication initiation based on the mechanism of protein copy-number sensing. Using a mean-field approach, we first derive an analytical expression of the cell size at initiation based on three biological mechanistic control parameters for an extended initiator-titration model. We also study the stability of our model analytically and show that initiation can become unstable in multifork replication conditions. Using simulations, we further show that the presence of the conversion between active and inactive initiator protein forms significantly represses initiation instability. Importantly, the two-step Poisson process set by the initiator titration step results in significantly improved initiation synchrony with CV~1/N scaling rather than the standard 1/N scaling in the Poisson process, where N is the total number of initiators required for initiation. Our results answer two long-standing questions in replication initiation: (i) Why do bacteria produce almost two orders of magnitude more DnaA, the master initiator proteins, than required for initiation? (ii) Why does DnaA exist in active (DnaA-ATP) and inactive (DnaA-ADP) forms if only the active form is competent for initiation? The mechanism presented in this work provides a satisfying general solution to how the cell can achieve precision control without sensing protein concentrations, with broad implications from evolution to the design of synthetic cells.
                Cover page: Bacterial Replication Initiation as Precision Control by Protein Counting.
                • Article
                • Peer Reviewed
                UC San Diego Previously Published Works (2021)
                We examine five quantitative models of the cell-cycle and cell-size control in Escherichia coli and Bacillus subtilis that have been proposed over the last decade to explain single-cell experimental data generated with high-throughput methods. After presenting the statistical properties of these models, we test their predictions against experimental data. Based on simple calculations of the defining correlations in each model, we first dismiss the stochastic Helmstetter-Cooper model and the Initiation Adder model, and show that both the Replication Double Adder (RDA) and the Independent Double Adder (IDA) model are more consistent with the data than the other models. We then apply a recently proposed statistical analysis method and obtain that the IDA model is the most likely model of the cell cycle. By showing that the RDA model is fundamentally inconsistent with size convergence by the adder principle, we conclude that the IDA model is most consistent with the data and the biology of bacterial cell-cycle and cell-size control. Mechanistically, the Independent Adder Model is equivalent to two biological principles: (i) balanced biosynthesis of the cell-cycle proteins, and (ii) their accumulation to a respective threshold number to trigger initiation and division.
                  Cover page: Quantitative Examination of Five Stochastic Cell-Cycle and Cell-Size Control Models for Escherichia coli and Bacillus subtilis
                  • Article
                  • Peer Reviewed
                  UC San Diego Previously Published Works (2018)
                  Bacterial physiology is a branch of biology that aims to understand overarching principles of cellular reproduction. Many important issues in bacterial physiology are inherently quantitative, and major contributors to the field have often brought together tools and ways of thinking from multiple disciplines. This article presents a comprehensive overview of major ideas and approaches developed since the early 20th century for anyone who is interested in the fundamental problems in bacterial physiology. This article is divided into two parts. In the first part (sections 1-3), we review the first 'golden era' of bacterial physiology from the 1940s to early 1970s and provide a complete list of major references from that period. In the second part (sections 4-7), we explain how the pioneering work from the first golden era has influenced various rediscoveries of general quantitative principles and significant further development in modern bacterial physiology. Specifically, section 4 presents the history and current progress of the 'adder' principle of cell size homeostasis. Section 5 discusses the implications of coarse-graining the cellular protein composition, and how the coarse-grained proteome 'sectors' re-balance under different growth conditions. Section 6 focuses on physiological invariants, and explains how they are the key to understanding the coordination between growth and the cell cycle underlying cell size control in steady-state growth. Section 7 overviews how the temporal organization of all the internal processes enables balanced growth. In the final section 8, we conclude by discussing the remaining challenges for the future in the field.
                    Cover page: Fundamental principles in bacterial physiology—history, recent progress, and the future with focus on cell size control: a review
                    • Article
                    • Peer Reviewed
                    UC San Diego Previously Published Works (2014)
                    At all but the slowest growth rates, Escherichia coli cell cycles overlap, and its nucleoid is segregated to daughter cells as a forked DNA circle with replication ongoing-a state fundamentally different from eukaryotes. We have solved the chromosome organization, structural dynamics, and segregation of this constantly replicating chromosome. It is locally condensed to form a branched donut, compressed so that the least replicated DNA spans the cell center and the newest DNA extends toward the cell poles. Three narrow zones at the cell center and quarters contain both the replication forks and nascent DNA and serve to segregate the duplicated chromosomal information as it flows outward. The overall pattern is smoothly self-replicating, except when the duplicated terminus region is released from the septum and recoils to the center of a sister nucleoid. In circular cross-section of the cell, the left and right arms of the chromosome form separate, parallel structures that lie in each cell half along the radial cell axis. In contrast, replication forks and origin and terminus regions are found mostly at the center of the cross section, balanced by the parallel chromosome arms. The structure is consistent with the model in which the nucleoid is a constrained ring polymer that develops by spontaneous thermodynamics. The ring polymer pattern extrapolates to higher growth rates and also provides a structural basis for the form of the chromosome during very slow growth.
                      Cover page: The multifork Escherichia coli chromosome is a self-duplicating and self-segregating thermodynamic ring polymer
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