Bulletin of the American Physical Society
APS March Meeting 2021
Volume 66, Number 1
Monday–Friday, March 15–19, 2021; Virtual; Time Zone: Central Daylight Time, USA
Session X13: Physics in Synthetic BiologyLive
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Sponsoring Units: DBIO DSOFT Chair: Gabor Balazsi, State Univ of NY - Stony Brook; Guillaume Lambert, Cornell University |
Friday, March 19, 2021 8:00AM - 8:12AM Live |
X13.00001: Crowding-induced spatial organization of gene expression in cell-sized vesicles Gaurav Chauhan, Elizabeth Norred, Michael Simpson, Steven M Abel A major limitation of cell-free expression systems is the lack of a means to spatially organize gene expression components to mimic cellular environments. We used computer simulations to guide experimental efforts to control the spatial organization of DNA and ribosomes in cell-sized vesicles using macromolecular crowding. With a coarse-grained model of DNA plasmids and crowders, we showed that plasmids were uniformly distributed at low levels of crowding but, due to depletion interactions, became strongly adsorbed to confining surfaces at high levels of crowding. We validated these results using fluorescently-labelled DNA plasmids and ribosomes in cell-sized vesicles. At large concentrations of the crowding agent Ficoll 70, DNA plasmids preferentially localized near vesicle membranes while ribosomes remained uniformly distributed. We then used kinetic Monte Carlo simulations and a coupled mRNA/protein reporter technique to understand the dynamics of transcription and translation. Crowding-induced localization of DNA to vesicle surfaces resulted in lower protein abundance and decreasing translational efficiency with increasing system size. Our approach demonstrates a cell-free platform that provides a means to better understand spatial control of gene expression. |
Friday, March 19, 2021 8:12AM - 8:24AM Live |
X13.00002: Synthetic biological tuning of metastasis regulator reveals nonmonotonic phenotype response Yiming Wan, Joseph Cohen, Kevin Farquhar, Nicholas Nest, Alex smashnov, Mariola Szenk, Gabor Balazsi Dysregulation of transcription factor (TF) networks is a major pathogenic event in metastasis - one of the leading factors associated with high mortality of cancer. Although many qualitative relationships between oncogenic regulators and cancer phenotypes are known, their quantitative characterization and understanding is lacking. We established a synthetic biological framework to enable such quantitative characterization, and applied it to BACH1, a metastasis regulator. Upon establishing and characterizing BACH1 control in stable monoclonal in triple-negative breast cancer cell lines, we uncovered a non-monotonic relationship between BACH1 expression and cell invasion ability. We confirmed and characterized this non-monotonic landscape by analyzing BACH1 expression histograms of the invaded population at critical induction levels. A single-cell-level model explained the observed histogram shifts. Overall, we propose a systematic approach to quantify dose-dependent relationships between TFs and their phenotypic/regulatory functions in mammalian cells, with relevance to cancer, tissue homeostasis and development. |
Friday, March 19, 2021 8:24AM - 8:36AM Live |
X13.00003: Monitor, categorize and manipulate label-free water-in-oil droplets in microfluidic systems Tobias Neckernuss, Christoph Frey, Jonas Pfeil, Daniel Geiger, Ilia Platzman, Othmar Marti, Joachim Spatz A key point of droplet based microfluidics is the availability of powerful but easy-to-implement methods for high throughput real-time analysis and automated manipulation of the droplets. We developed a novel optical device, consisting of a fast camera with integrated data processing for smart and fast algorithms enabling label-free real-time monitoring and active manipulation of passing droplets. The device continuously analyzes up to 3000 particles per second in real-time with respect to bright-field image parameters like size, brightness, granularity, circumference, speed and many more. According to these parameters and combinations thereof, the passing droplets can be sorted. We measure different droplet production parameters and demonstrate label-free detection of cells encapsulated in droplets. Furthermore, we performed label-free sorting of cell laden droplets from empty droplets. The peripheral sorting electronics are controlled by our device. Decision making is based on predefined parameter ranges that are compared to the measurement results of the droplets right before the sorting gate. Similarly, in another experiment we demonstrate efficient sorting of droplets depending on size. |
Friday, March 19, 2021 8:36AM - 8:48AM Live |
X13.00004: Improved CRISPRi gene circuit function via context-sensitive antisense RNA sequestration David Specht, Guillaume Lambert By using the binding of the catalytically-dead CRISPR protein dCas12a, we can create programmable gene circuit elements in E. coli. These ‘CRISPRgates’ are simple NOT gate elements which can target genes or other CRISPRgate elements and in principle can be combined to create complex genetic circuits, a fundamental goal of synthetic biology. While natural transcription factors have built-in advantages for transcriptional regulation (e.g. cooperativity), they are not programmable or individually orthogonal and a limited number of them exist. Repression with CRISPR is advantageous because we can in principle repress many different targets simultaneously without crosstalk. However, such gene circuit elements behave poorly when placed in series due to signal loss that occurs due to leaky repression (e.g. NOT NOT NOT != NOT) and retroactivity effects due to a shared pool of Cas proteins. By utilizing antisense RNAs to sequester guide RNA transcripts in combination with appropriate spatial arrangement of gene circuit nodes, we demonstrate a mechanism to suppress leaky CRISPRi repression and restore logical gene circuit function when elements are used in series. |
Friday, March 19, 2021 8:48AM - 9:00AM Live |
X13.00005: Emergence of homochirality in large molecular systems Gabin Laurent, David Lacoste, Pierre Gaspard
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Friday, March 19, 2021 9:00AM - 9:12AM Live |
X13.00006: Control of Gene Expression through Control of Plasmid Copy Number Miles Rouches, Guillaume Lambert As mobile genetic elements capable of self-replication, plasmids are perhaps the most versatile and widely used tool in synthetic biology. Though we have a clear picture of the elegantly evolved mechanisms governing the control of plasmid replication our understanding of the effects of plasmid copy number on gene expression and host physiology is vague. In this work we demonstrate robust control of plasmid copy number in ColE1 plasmids in several manners – using inducible promoters and massively parallelized assays to investigate the parameters governing plasmid copy number. We leverage this control to better understand the effect of plasmid copy number on host cell processes. The effects of copy number on gene expression and their entailed effects on host cell growth are investigated as are the effect of gene copy number on the efficiency of CRISPRi-based repression. We further use our control of plasmid copy number to investigate the dual perils that necessitate control of plasmid copy number in the first place - plasmid loss and metabolic burden, casting light onto the limits of plasmid replication. |
Friday, March 19, 2021 9:12AM - 9:48AM Live |
X13.00007: Footprints in the noise: understanding gene circuit structure and function using noise spectroscopy Invited Speaker: Michael Simpson The study of gene expression noise has a long history with analytical roots in the early 1900s and biological roots that extend at least back to Delbruck’s work in the 1940s. More recent work, with gene regulation conceptualized in terms analogous to electronic circuits, has elucidated the direct connection between the “structure” of the noise and the structure and function of the underlying gene circuits. This talk will describe the history of gene expression noise spectroscopy as a means to probe gene regulatory arrangements such as positive and negative autoregulation, including the positive and negative feedback loops in the HIV gene circuit. The talk will conclude with a focus on more recent work to use expression noise spectroscopy to understand the synergistic effects of confinement and macromolecular crowding in cell-free expression systems. |
Friday, March 19, 2021 9:48AM - 10:00AM Live |
X13.00008: Characterization of programmable CRISPR-based toggle switches in Escherichia coli Yasu Xu, Guillaume Lambert Recent developments in CRISPR-Cas systems have ushered a new generation of powerful genetic engineering tools in synthetic biology. In particular, a catalytically ‘dead’ version of Cas proteins that lack nuclease activity can essentially function as a logic NOT gate by selectively binding to a promoter and preventing transcription initiation. This work aims to provide an efficient and systematic workflow to design, assemble and fine-tune CRISPRi based genetic toggle switches from many potential constructs. By assuming that CRISPRi turns off transcription shares the same mechanism as simple repression motif, a thermodynamic model is developed to investigate the interaction of two mutually repressed CRISPRi NOT gate under various cellular background by tuning the availability and affinity of specific binding sites, competing sites, and the activity of targeted promoter. Next, a rapid and versatile characterization platform, X-seq, was used to assemble and compare hundreds and thousands of potential constructs in parallel, from which a few candidates can be picked for further fine-tuning. In the end, the cooperativity of chosen toggle switch candidates can be optimized through pairing with a proper competing sites number that is loaded on a helper plasmid with a tunable copy number. |
Friday, March 19, 2021 10:00AM - 10:12AM Live |
X13.00009: Improving CRISPR gene circuit design using Physics and Synthetic Biology Guillaume Lambert, David Specht, Yasu Xu The versatility of CRISPR-Cas endonucleases as a tool for synthetic biology has lead to diverse applications beyond gene editing that include programmable transcriptional control and nucleic acid detection. Most CRISPR-Cas systems, however, suffer from off-target effects and unpredictable non-specific binding that negatively impact their reliability and broader applicability. To better evaluate the impact of mismatches on DNA target recognition and binding for CRISPR-Cas variants, we develop a massively parallel CRISPR interference assay to measure the binding energy between tens of thousands of target sequences. Using a general thermodynamic model of CRISPR-Cas binding dynamics, we unravel the complex energetic landscape of several CRISPR-Cas systems. Our approach provides a mechanistic understanding of target recognition and DNA binding of CRISPR-Cas variants, which should contribute to the advancement of recent synthetic biology efforts to repurpose dCas as gene circuit elements that behave orthogonally and operate independently without crosstalk. |
Friday, March 19, 2021 10:12AM - 10:48AM Live |
X13.00010: Synthetic Biology to Endow Novel Sensing and Reporting Functions in Plants Invited Speaker: C. Neal Stewart Plant biotechnology has made great headway towards engineering plants for new agronomic functions, but precise gene expression of transgenes remains a challenge. Synthetic biology, which combines design and computational tools to create novel DNA sequence for engineering, holds the key to drastically improve the control of gene expression. The Center for Agricultural Synthetic Biology was recently founded to apply synthetic biology toward agricultural organisms. The presentation will focus on gains to produce novel plants to detect and report environmental contaminants. The key technological gain is design, building, and testing synthetic promoters in a high-throughput manner. |
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