Bulletin of the American Physical Society
APS March Meeting 2023
Volume 68, Number 3
Las Vegas, Nevada (March 5-10)
Virtual (March 20-22); Time Zone: Pacific Time
Session N08: Synthetic BiologyFocus
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Sponsoring Units: DBIO Chair: Jonathan Michel, Rochester Institute of Technology; Sahand Rahi, Ecole Polytechnique Federale de Lausanne Room: Room 131 |
Wednesday, March 8, 2023 11:30AM - 11:42AM |
N08.00001: Stochastic Model and Optimization of SELEX Yue Wang Antibodies are important biomolecules that are often designed to recognize target antigens. However, they are expensive to produce and their relatively large size prevents their transport across lipid membranes. An alternative to antibodies is aptamers, short (~ 15-60 bp) oligonucleotide (and amino acid sequences) with specific secondary and tertiary structures that govern their affinity to specific target molecules. Aptamers are typically generated via solid phase oligonucleotide synthesis before selection and amplification through Systematic Evolution of Ligands by EXponential enrichment (SELEX), a process based on competitive binding that enriches the population of certain strands while removing unwanted sequences, yielding aptamers with high specificity and affinity to a target molecule. Mathematical analyses of SELEX have been formulated in the mass action limit, which assumes large system sizes and/or high aptamer and target molecule concentrations. In this paper, we develop a fully discrete stochastic model of SELEX. While converging to a mass-action model in the large system-size limit, our stochastic model allows us to study statistical quantities when the system size is small, such as the probability of losing the best-binding aptamer during each round of selection. Specifically, we find that optimal SELEX protocols in the stochastic model differ from those predicted by a deterministic model. |
Wednesday, March 8, 2023 11:42AM - 11:54AM |
N08.00002: Crowd control: Organizing gene expression in cell-sized vesicles with macromolecular crowding Gaurav Chauhan, Elizabeth Norred, Michael L Simpson, Steven M Abel In cells, gene expression is regulated in part by the spatial organization of transcription and translation. Unfortunately, current cell-free approaches are unable to control the organization of molecular components needed for gene expression, which limits the ability to probe and utilize its effects. Here we show, using computational and experimental approaches, that macromolecular crowding can be used to control the spatial organization and translational efficiency of gene expression in cell-sized vesicles. Computer simulations and imaging experiments reveal that, as crowding is increased, DNA plasmids become localized at the inner surface of vesicles. Ribosomes, in contrast, remain uniformly distributed. We further carried out cell-free protein synthesis reactions in cell-sized vesicles and quantified mRNA and protein abundance. At sufficiently high levels of crowding, we observed localization of mRNA near vesicle surfaces, a decrease in translational efficiency and protein abundance, and anomalous scaling of protein abundance as a function of vesicle size. Mathematical modeling demonstrates that these results are consistent with high levels of crowding causing altered spatial organization and slower diffusion. Our work demonstrates a straightforward way to control the organization of gene expression in cell-sized vesicles and provides insight into the spatial regulation of gene expression. |
Wednesday, March 8, 2023 11:54AM - 12:06PM |
N08.00003: An inducible and titratable site-specific in vivo recombination system in Escherichia coli Elisa Garabello, Andrea Giometto Site-specific recombination systems, such as Cre-lox, have notably enhanced spatial and temporal control of gene expression, and cell labeling techniques like the Brainbow method. Some applications, such as biosensors, might benefit from in vivo recombination events at low rates that are tunable with an external inducer. Developing such a system is challenging due to the high efficiency of DNA recombinases. In Saccharomyces cerevisiae, this has been obtained by fusing the Cre recombinase to the estradiol-binding domain (EBD) which sequesters the Cre in the cytoplasm until estradiol is provided. Once the Cre is in the nucleus, it is able to cleave DNA between two lox sites located on the genome. The development of tunable, low-rate recombination systems for prokaryotes has been very limited, and achieved only through light-inducible systems. We developed and characterized a Cre/lox system that allows us to perform in vivo recombineering at tunable, low rates in Escherichia coli, using chemically inducible promoters with low leakage and a degradation tag on the Cre recombinase. This system can be introduced on either plasmids or the E. coli chromosome. Future applications include controlled and reproducible cell conversion in evolutionary experiments and the development of recombination-based biosensors. |
Wednesday, March 8, 2023 12:06PM - 12:18PM |
N08.00004: Atomic Force Microscopy experiment and Monte-Carlo study of the elasticity of exosomes Vikash Pandey, Hanna Kylhammar, Vipin Agrawal, Fredrik Stridfeldt, Apurba Dev, Dhrubaditya Mitra Nano-vesicles (50-300 nm) are ubiquitous in biology. Among them, extra-cellular |
Wednesday, March 8, 2023 12:18PM - 12:30PM |
N08.00005: Excitation and inhibition imbalance affects dynamical complexity through symmetries mathieu ouellet, Jason Z Kim, Harmange Guillaume, Sydney M Shaffer, Lee Bassett, Dani S Bassett From the perfect radial symmetries of radiolarian mineral skeletons to the broken symmetry of homochirality, the logic of Nature's regularities has fascinated scientists for centuries. Some of Nature's symmetries are clearly visible in morphology and physical structure, whereas others are hidden in the network of interactions among system components. Just as visible symmetries and asymmetries contribute to the system's beauty, might hidden symmetries contribute to the system's functional harmony? And if so, how? Here we demonstrate that the interaction networks of biological systems---from cell signaling to cancer---display a form of dynamical reflection symmetry that serves to expand their dynamical complexity. The expansion proceeds according to precise rules regarding the lengths of dynamical cycles, made possible by a peculiar imbalance between excitation and inhibition. To probe the conditions under which this reflection symmetry evolves, we use a multi-objective genetic algorithm to produce networks with long dynamical cycles. We find that local structural motifs that break the reflection symmetry are deleted in the evolutionary process, providing evidence for symmetry's causal role in dynamical complexity. Finally, we identify symmetries in continuously valued scRNA-seq data of cancer cells, and show that these symmetries can be used to classify drug-responsive versus drug-resistant cells. Broadly, our work reveals a class of hidden symmetries in biology, present in the network of interactions among system components, and it underscores their functional importance. Mathematically simple and computational inexpensive, our approach is applicable to the types of biological data commonly acquired in modern experiments. |
Wednesday, March 8, 2023 12:30PM - 12:42PM |
N08.00006: Microbial Division of Labor for Mixed Sugar Fermentation Siqi Liao, Ting Lu The feedstocks of microbial fermentation are usually present in a form of mixed substrates. Full utilization of such mixtures can be achieved by a 'superbug' strain capable of simultaneous multi-substrate consumption or, alternatively, by a consortium of strains through a division of labor (DOL) whereby each member specializes in utilizing one specific substrate. Conceptually, the latter allows to reduce the high metabolic burden and pathway crosstalk, thus promising a higher fermentation efficiency and a better overall productivity. However, it remains unclear whether DOL indeed offers these advantages over single strains and under what circumstances DOL is superior for mixed substrate fermentation. In this study, we develop a theoretical framework for microbial mixed-sugar fermentation and use it to compare the performances of the superbug and DOL strategies in chemostats. Through the analysis from a minimal model to systems with increasing complexity, we find that the conditions favorable to DOL can be specified by a general rule regarding the metabolic burden and pathway crosstalk effect of superbug, inflow concentrations of substrates, and dilution rate of the chemostat. Our work provides quantitative insights into the design principles of microbial consortia for multi-substrate fermentation. |
Wednesday, March 8, 2023 12:42PM - 12:54PM |
N08.00007: Molecular analysis of the type III interferon complex and its applications in protein design and engineering William S Grubbe, Fabian Byléhn, Walter Alvarado, Juan L Mendoza, Juan J De Pablo Type III interferons (IFNL1-4) are proteins with critical roles in the immune system and signal only at barrier tissues, making them attractive therapeutic candidates. However, structural studies of the proteins and their signaling complexes have been limited by low-affinity protein-protein interactions and challenging expression conditions in the lab. While previous efforts have utilized extensive protein engineering to overcome these barriers, we use a combination of computational and experimental techniques to generate a detailed molecular analysis of the type III IFNs. Using molecular modeling and simulations, we quantify differences in residue contact and strain fluctuation, produce detailed free-energy landscapes, and reveal previously unknown structural features of the engineered and wild type extracellular signaling complexes; we then leverage this information to guide engineering at the receptor-receptor interface, increasing complex formation by as much as 2.5-fold and identifying hot spots of interactions between the ligands and receptors. This two-pronged approach to protein design and engineering enables analysis of inaccessible protein complexes to generate molecular insights into IFNL behavior, elevating approaches to harness their therapeutic potential. |
Wednesday, March 8, 2023 12:54PM - 1:06PM |
N08.00008: Competitive Binding Facilitates Oscillations in a Non-Cooperative Repressilator Miles V Rouches, Louis B Cortes, Guillaume Lambert The repressilator is the simplest transcriptional regulatory network known to exhibit sustained oscillations. Deterministic models predict that oscillations are generally conditional to repressor cooperative binding. Here we present a repressilator made from deactivated CRISPR cas proteins, which, despite binding non-cooperatively to their target sites, nevertheless exhibit sustained oscillations over many cellular generations as observed in single cell microfluidics experiments. We explain how the cooperativity requirement can be circumvented by sequestration of repressors via competitive binding sites housed on a tunable copy number plasmid. These findings are explained through deterministic and stochastic models which are used to guide further inquiry into how the period, amplitude, and statistical properties of these oscillations can be controlled by competitive binding. |
Wednesday, March 8, 2023 1:06PM - 1:18PM |
N08.00009: Untangling the regulatory role of individual TFs in E. coli Robert C Brewster Predicting the effect of individual TFs on gene regulation can be challenging due to their entanglement with promoter specific effects such as TF-TF interactions, or network interactions (feedback, etc). To overcome this limitation, we have developed an approach using systematically designed synthetic gene targets in E. coli to quantify the pure regulatory function of individual TFs and to remove the “context dependent” entanglements of natural promoters. This methodology quantifies the level of regulation exerted on two distinct steps in the transcription process: the stabilization (or destabilization) of RNAP at the promoter and the rate of initiation of RNAP once bound. The magnitude of these two interactions depends on factors such as TF identity, binding position on the promoter and binding site sequence. These two parameters determine an individual TFs net regulatory effect on expression. To demonstrate this, we measured the regulatory characteristics of 30 unrelated TFs from a wide array of TF families and show that natural TFs regulate through a diverse combination of regulatory modes. Crucially, these two mechanisms do not function the same at every promoter indicating that the net regulatory function of a TF is promoter dependent. To demonstrate this principle, we further measured the regulation of these TFs on a library of promoters of different strength and find specific TFs work better on strong promoters rather than weak ones and vice versa. Overall, we find that by distinguishing the regulatory modes of individual TFs we can determine the effectiveness of specific TFs on different promoters. |
Wednesday, March 8, 2023 1:18PM - 1:54PM Author not Attending |
N08.00010: David R. Liu (Harvard University) Invited Speaker: David R Liu
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