Bulletin of the American Physical Society
APS March Meeting 2022
Volume 67, Number 3
Monday–Friday, March 14–18, 2022; Chicago
Session F04: Synthetic Biology IIFocus Recordings Available
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Sponsoring Units: DBIO GSNP Chair: Guillaume Lambert, Cornell University Room: McCormick Place W-176C |
Tuesday, March 15, 2022 8:00AM - 8:12AM |
F04.00001: Friend or Foe: The Potential of Transposable Element Tuning Davneet Kaur, Thomas E Kuhlman Transposable elements (TEs) are mobile DNA sequences that can be cut or copied and reintegrated elsewhere into the genome. Long considered junk DNA, owing to their disruptive frolicking, TEs have instead been determined to be major contributors to disease, development, and evolution. The evolutionary arms race between TEs and the DNA modification systems that curb the damage of TE proliferation has created a delicate balance of TE regulation in our cells. However, this tight control of TE propagation loosens over the course of our lives. The deregulation of TEs has been associated with the onset of several developmental diseases and human cancers. On the other hand, increased TE activity in cancerous cells in blind mole rats has been shown to impart them with cancer resistance by inducing concerted cell death of diseased cells. The difference in these antithetical outcomes is the tuning of TE activity, demonstrating the importance of characterizing TE dynamics. The potential benefits are twofold: in the prevention of TE-associated diseases and the promise of developing TEs as genetic therapeutics for diseases such as cancers. In this study we characterize the dynamics of the autonomous TE, IS608. Several questions about IS608's transposition process have remained unanswered. Firstly, an unknown mechanism is employed to resolve the TE-complementary strand which may or may not result in TE replication. Secondly, IS608 contains a gene of unknown function, TnpB. We address these questions through real-time tracking of fluorescence-tagged genetic sequences, that allow us to measure transposase, TnpB protein and excision levels in live cells. We show that TnpB works with transposase to increase the active TE count, not through replication but through conservation of active TEs. We propose a model in which TnpB increases the efficiency of successful reinsertion of excised TEs. TnpB may achieve this by use of its RuvC-like domain to improve TE insertion. |
Tuesday, March 15, 2022 8:12AM - 8:24AM |
F04.00002: A Plasmid System with Tunable Copy Number Miles V Rouches, Louis B Cortes, Yasu Xu, Guillaume Lambert Plasmids are one of the most commonly used and time-tested molecular biology platforms for synthetic biology and recombinant gene expression in bacteria. Despite their ubiquity, little consideration is given to metabolic effects and fitness costs of plasmid copy numbers on engineered genetic systems. Here, we demonstrate two methods we've developed that allow for finely-tuned control of ColE1 plasmid copy number: a plasmid with an anhydrotetracycline-inducible copy number, and a massively parallel assay that is used to generate a continuous spectrum of ColE1-based copy number variants. These systems are used to probe the effects of plasmid and gene copy number on cellular growth rates, gene expression, biosynthetic pathway production, and the behavior of a simple genetic circuit. We perform single-cell timelapse measurements to characterize plasmid loss, runaway plasmid replication, and quantify the impact of plasmid copy number on variability in gene expression. Using our massively parallel assay, we find that plasmids impose a linear metabolic burden on their hosts, hinting at a simple relationship between metabolic burdens and plasmid DNA synthesis. Our plasmid system with tunable copy number should allow for a precise control of gene expression and highlight the importance of tuning plasmid copy number as a tool for the optimization of synthetic biological systems. |
Tuesday, March 15, 2022 8:24AM - 8:36AM |
F04.00003: Developing A Genetic Toggle Switch in Arabidopsis Tessema K Kassaw, Wenlong Xu, Christopher S Zalewski, Katherine A Kiwimagi, Ron Weiss, Mauricio S Antunes, Ashok Prasad, June I Medford Plant synthetic biology has significant technological potential, but the complexity of plant biology has made it difficult to engineer complex information-processing circuits into plants. The synthetic genetic toggle switch, which translates binary external stimuli into binary internal outcomes, was the earliest information-processing synthetic circuit developed in E. Coli in 2000. Here we report on results from a long collaborative effort to develop the first synthetic toggle switch in Arabidopsis. We used a forward engineering approach by first analyzing synthetically designed repressors in plant protoplasts[1]. Parameters obtained allowed us to predict the combinations most likely to work in the plant. We then chose two combinations to test, assembled and inserted them in Arabidopsis and used luciferase luminescence to report on circuit behavior. Assessing circuit behavior in plants was affected by unexpected phenomena such as tissue specific differences and the confounding effects of plant growth. Despite these issues we were able to successfully fit a mathematical model to the plant data using MCMC sampling. Analysis of the results led to the conclusion that one of the two combinations satisfied the tests for bistability. This circuit is, to our knowledge, the first synthetic genetic toggle switch in a plant[2]. |
Tuesday, March 15, 2022 8:36AM - 8:48AM |
F04.00004: Noise and tunability of a programmable CRISPR platform for gene network regulation Yiming Wan, Rafal Krzyszton, Joseph Cohen, Damiano Coraci, Gabor Balazsi Viral infections and cancer often manifest by transcriptome dysregulation in human cells. CRISPR-Cas systems could provide ways to correct transcriptome dysregulation, thereby supplementing our native immunity against viral infection, tumorigenesis and metastasis. However, the benefits and deleterious effects of such CRISPR systems could depend on the level and noise of Cas expression, which are currently unknown. Here, we adapted a negative-feedback synthetic gene circuit to precisely regulate the Cas13d effector expression. Using host cell genome engineering approaches, we integrated site-specifically Cas13d-tuning constructs into a safe harbor site in the human genome via recombinase-mediated cassette exchange. We compare gene expression data with computational models to understand the transcript-tuning capability of Cas13d-containing synthetic gene circuits. Additionally, we develop orthogonally integrated on/off switches of guide RNA expression, making the full system programmable for regulation of arbitrary human or viral genes. By targeting SARS-CoV-2 gene fragments we found dose-responsive reduction of expression, suggesting a potential for regulated viral defense systems with minimal side effects. |
Tuesday, March 15, 2022 8:48AM - 9:00AM |
F04.00005: Factors that affect the bistability of programmable CRISPR-based toggle switches in Escherichia coli Yasu Xu Genetic toggle switches, as one of most basic genetic circuits, is the building block for more advanced circuits with many practical applications in various fields. However, currently widely used version that based on limited pairs of promoter-repressor pairs suffers from low orthogonality and programmability. Recent progress in CRISPR-Cas systems have shown its potential as a new generation of genetic editing tool, especially, a catalytically ‘dead’ version of Cas proteins that lack nuclease activity can essentially function as a logic NOT gate by programing the complex binding to a promoter to interfere transcription. This work aims to provide an systematical workflow that simulate, assemble and sort out bistable CRISPRi based toggle switches from thousands of potential constructs.We first developed a thermodynamic model to investigate parameters,such as binding sites affinity and availability, and targeted promoter activity on that affects the bistability of a toggle switch. Next, a versatile X-Seq assay is used to investigate each of these parameters by characterizing many potential constructs in parallel. We found that by carefully matching promoter strength and the copy number of toggle swtich construct, we are able to see bistabilty in the cell. |
Tuesday, March 15, 2022 9:00AM - 9:12AM |
F04.00006: Computational Investigation of Cell Shape Changes Driven by Actomyosin Contractility Fahmida Sultana Laboni, Makito Miyazaki, Taeyoon Kim Changes in cell shapes are mostly driven by forces generated from the cytoskeleton. Recently, several in vitro experiments developed a synthetic cell-like system consisting of actin networks, cross-linking proteins, and myosin motors encapsulated by lipid vesicles or water droplets in order to study cell-scale behaviors facilitated by forces generated from actomyosin networks. However, these experiments are very hard to conduct, thus preventing exploration of wide parametric spaces. To overcome experimental limits and thus perform extensive parametric studies, we developed a novel agent-based model for a minimal cell-like structure comprised of discrete actomyosin cortex, osmotic pressure, and cell membrane simplified into a triangulated mesh. The cortex is coupled to the cell membrane to various extents via cross-linking proteins. Using this model, we found how cell shapes are regulated by a competition between actomyosin contractility that tends to induce contraction and osmotic pressure that tends to lead to expansion. We demonstrated that bulge that mimics cell blebs can be formed when the coupling level between the cortex and the membrane is intermediate. Our results provide insights into understanding how cell shapes are regulated under diverse conditions. |
Tuesday, March 15, 2022 9:12AM - 9:48AM |
F04.00007: Identification of pathological nodes in the protein-protein interaction network maps of triple negative breast cancer Invited Speaker: Nandakumar Yellapu Advances in sequencing technologies have facilitated the identification of molecular targets for numerous human diseases, including cancer. Protein-protein interaction (PPI) networks derived from large-scale molecular data sets provide a global picture of the cellular process underlying pathogenesis and disease progression. As is typical in PPI networks, there are a few proteins that are connected with other proteins with high degree acting as hub or hot-spot nodes. The dysfunction of such hot-spot nodes is associated with disease development and progression. Proteins have either single or multi reactive interfaces and studying the interface properties of cancer-related proteins promises to help elucidate their role in PPI networks. In this study we analyzed PPI networks in Triple Negative Breast Cancer (TNBC) to delineate the hot-spot nodes and the related reactive protein interfaces. We provide a detailed analysis of TNBC-related PPI interfaces and the topological properties of the network. Cancer-related proteins have a defined unique binding interface, which may indicate their specificity and affinity for the cancer-related interactions in the networks. In addition, cancer related proteins in the network tend to interact with their partners through distinct interfaces that are crucial for cellular pathogenicity. Such proteins form the nodes with higher essentiality in the network. The findings from this study point to new targets in the development of TNBC as well as the key binding surfaces of putative drug candidates. |
Tuesday, March 15, 2022 9:48AM - 10:00AM |
F04.00008: CRISPR-mediated imaging of three-dimensional genomic loci in live cells Yanyu Zhu, Mengting Han, Xueqiu Lin, Haifeng Wang, Lei Stanley Qi The three-dimensional (3D) organization and dynamics of eukaryotic genome play significant roles in regulating gene transcription and cellular function. Conventional CRISPR-based imaging approach is powerful for imaging specific chromatin regions in live cells but relies on laborious genome engineering. We recently developed a versatile imaging technique, LiveFISH, which visualizes genomic element dynamics in live cells by delivering in vitro assembled fluorescent ribonucleoproteins (fRNPs) of fluorophore-labeled guide RNAs and purified nuclease-deactivated dCas9. However, it was mostly applicable to imaging repetitive genomic regions. Here we report an improved LiveFISH method that could target non-repetitive genomic loci of choice. Our method integrates computational guide RNA probe design, chemical fluorophore labeling, and high-quality cell delivery platforms. We characterized quantitively the dependence of imaging quality on a series of probe design parameters and applied this improved LiveFISH technology to track 3D dynamics and organization of enhancers and promoters of different genes with high localization precision. The platform can work potentially in diverse cell types to study the causality between the 3D genome and gene regulation. |
Tuesday, March 15, 2022 10:00AM - 10:12AM |
F04.00009: Synthetic biology tools to create electroactive biofilms James A Boedicker Many bacterial species are naturally capable of extracellular electron transfer, leading to long-distance electron transport and the formation of electrically conductive biofilms. Shewanella oneidensis is one such exoelectrogenic organism, which uses a network of multiheme c-type cytochromes to transfer electrons from the cellular interior to external surfaces, including adjacent cells. Synthetic biology tools to manipulate such exoelectrogens are largely lacking, especially when compared to the toolbox available for more traditional laboratory strains. To address this need, we have adapted several synthetic biology constructs to enable light-induced gene expression within the bacteria Shewanella oneidensis. Light was used to regulate the expression of genes involved in cell-cell adhesion and biofilm formation. An adhesion protein native to another species of bacteria was identified that led to light-controlled patterning of biofilms. Such biofilms were thicker and more uniform than wildtype biofilms formed by S. oneidensis and exhibited extracellular redox activity similar to the wildtype strain. Using these tools, conductive biofilms could be patterned on surfaces with controlled geometry and placement. The engineered strains were used to characterize the intrinsic conductivity of living S. oneidensis biofilms and demonstrate new applications involving the precise placement of electrically active biofilms on electronic devices. |
Tuesday, March 15, 2022 10:12AM - 10:24AM |
F04.00010: AI-based analysis of microbial ecological dynamics Shangying Wang, Simone Bianco Microbial ecology studies the interactions, population dynamics, and distributions of microbes and their interaction with the environment. Understanding the mechanisms controlling community diversity and functions is an important, but poorly understood, topic in ecology, particularly in microbial ecology. The functions performed by microbial communities are shaped by complex and dynamic interactions between constituent community members and their environment. The bottom-up construction of synthetic microbial community enables the investigation of reduced complexity assemblages with control of initial community composition. However, this is still extremely difficult with the increased number of microbial species. In addition, the stochastic property of the biological systems makes the problem more complicated. Even microbial communities with identical initial abundances of constituent community members in identical growth environments, can demonstrate diverse functions. In this work, we develop an AI-based approach that can predict the conditional probability distribution of a quantitative function/behavior with a limited number of observations per input condition (initial composition and the environment factors). Our work enables the rational design of synthetic microbial communities with possible applications in health, agriculture, and bioprocessing. |
Tuesday, March 15, 2022 10:24AM - 10:36AM Withdrawn |
F04.00011: Dynamical Model for a Bistable Contractile Molecular Injection System Noah Toyonaga Contractile Injection Systems (CIS) are biomolecular mechanisms used by viruses to puncture cells prior to infection. CIS consist of a stiff penetrating tube bound to a sixfold symmetric sheath consisting of a stack of identical ring-like structures. During injection this sheath transitions between an extended and contracted configuration. The injection is triggered by a localized conformational change in the baseplate which initiates a contraction wave in the sheath that propagates from the baseplate to the distal end. Drawing from recent structural evidence for the conformation changes at the molecular level, we model this molecular machine using a simplified geometry of inextensible filaments inspired by chebyshev nets. This geometry is fully described by a single angle that varies as a function of space and time. We show that the interaction of the bending of the chebyshev fibres and a local bistability of the sub-unit allows us to recover the contraction wave observed in computational and biological experiments, and validate our model by building mechanisms at the desktop scale. We further use our model to estimate the dynamics and forces observed during contraction that may be directly measured in the biological setting. |
Tuesday, March 15, 2022 10:36AM - 10:48AM |
F04.00012: Inferring gene regulation from static snapshots of gene expression variability Euan Joly-Smith, Jerry Zitong Wang, Fotini Papazotos, Paige Allard, Laurent Potvin-Trottier, Andreas Hilfinger A key challenge of systems biology is to translate cell heterogeneity data obtained from single-cell sequencing or flow cytometry experiments into causal and dynamic interactions. We show how static population snapshots of gene expression reporters can be used to infer causal and dynamic properties of gene regulatory networks without using perturbations. For instance, we derive correlation conditions that detect causal interactions and closed-loop feedback regulation in gene regulatory networks from snapshots of transcript-levels. Furthermore, we show how oscillating transcription rates can be identified from the variability of co-regulated fluorescent proteins with unequal maturation times. Our approach exploits the fact that unequal fluorescent reporters effectively probe their upstream dynamics on separate time-scales such that their correlations implicitly encode information about the temporal dynamics of their upstream regulation. Synthetic genetic circuits provide exciting opportunities to verify these co-variability conditions with well characterized engineered systems. Lastly we report on ongoing experiments in which we quantitatively test our theory with variants of a synthetic oscillator, the Repressilator, in single-cells using time-lapse microscopy and microfluidics.
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