SynCell 2023

Aims of the conference

Registration

Travel funding

Program

The working groups
During the meeting, we will form ad-hoc working groups to develop short perspective papers on few key issues, roadblocks and opportunities in synthetic cell engineering.
The working group leaders will pitch their working group topics on Monday, at 11:15 - 12:00AM during the Working groups intro plenary session.
We invite you to join one of those working groups, to influence the perspectives paper on the topic that is closest related to your interests.
We hope that a collection of short, impactful papers coming out of this meeting will help to shape the directions of our field. 

Detailed program

Monday, May 22

Tuesday, May 23

Wednesday, May 24

Monday, May 22 sessions and keynotes

Monday, May 22, breakout sessions

Containers #1

Monday, May 22, Johnson room, 1:00PM - 2:30PM
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Beyond the Bench #1

Monday, May 22, Ski-U-Mah room, 1PM - 2:30PM
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Building Blocks #1

Monday, May 22, Swain room, 1PM - 2:30PM
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Containers #2

Monday, May 22, Johnson room, 3:00PM - 4:30PM
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Applications #1

Monday, May 22, Ski-U-Mah room, 3:00PM - 4:30PM
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Cytoskeleton #1

Monday, May 22, Swain room, 3:00PM - 4:30PM
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Building blocks #2

Monday, May 22, Johnson room, 4:45PM - 6:00PM
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Applications #2

Monday, May 22, Ski-U-Mah room, 4:45PM - 6:00PM
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Monday, May 22, keynote lectures
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Marileen Dogterom

Monday, May 22, Memorial Hall, 9:15AM - 10:00AM

A minimal cytoskeleton for synthetic cells

abstract

Avi Schroeder

Monday, May 22, Memorial Hall, 10:00AM - 10:45AM

Synthetic cells as platforms for drug delivery

Medicine is taking its first steps toward patient-specific cancer care. Nanoparticles have many potential benefits for treating cancer, including the ability to transport complex molecular cargoes, including siRNA and protein, as well as targeting specific cell populations.
The talk will explain the fundamentals of nanotechnology, from ‘barcoded nanoparticles’ that target sites of cancer where they perform a programmed therapeutic task. Specifically, liposomes diagnose the tumor and metastasis for their sensitivity to different medications, providing patient-specific drug activity information that can be used to improve the medication choice.
The talk will also describe how liposomes can be used for degrading the pancreatic stroma to allow subsequent drug penetration into pancreatic adenocarcinoma and how nanoparticle’ biodistribution and anti-cancer efficacy are impacted by the patient’s gender and, more specifically, the menstrual cycle.
The evolution of drug delivery systems into synthetic cells, programmed nanoparticles that have an autonomous capacity to synthesize diagnostic and therapeutic proteins inside the body, and their promise for treating cancer and immunotherapy, will be discussed.

Monday, May 22 poster session

Session Containers: liposome, microfluidics, coacervates etc. posters

Abbey Robinson

Abbey Robinson*, Jessica Lee, Anders Cameron, Christine D Keating, Katarzyna P. Adamala

poster number 10

Cell-free expressed membraneless organelles sequester RNA in synthetic cells

Compartments within living cells create specialized microenvironments, allowing for multiple reactions to be carried out simultaneously and efficiently. While some organelles are bound by a lipid bilayer, others are formed by liquid-liquid phase separation, such as P-granules and nucleoli1–3. Many of these membraneless condensates are formed by intrinsically disordered proteins (IDPs) that lack a discrete structure. LAF-1, an RNA helicase found in Caenorhabditis elegans P-granules, contains an intrinsically disordered region termed the RGG domain which plays a strong role in promoting phase separation4. Engineering compartments within synthetic cells facilitates the creation of more complex and multifaceted synthetic life-like systems. We describe a synthetic cell expressing phase-separating protein to form artificial membraneless organelles that are capable of sequestering RNA and reducing protein expression. We create complex microenvironments within synthetic cell cytoplasm, and introduce a tool to modulate protein expression in synthetic cells.

Anastassiya Schramm

Anastassiya Schramm*, Dr. Ilia Platzman, Prof. Dr. Joachim Spatz

poster number 13

Design of Stimuli-Responsive Multicompartment Vesicles for Immunological Applications

Programmed Death-Ligand 1 (PD-L1) together with its receptor, Programmed Cell-Death Protein 1 (PD-1) are crucial members of a PD-L1 - PD-1 checkpoint, which plays a major role in downregulating the immune system. A wide range of tumors has evolved to express PD-L1 on their surface, as well as on their exosomes in order to evade the immune system response. After prolonged interaction with this protein, T-cells become exhausted due to the continuous blockade of their PD-1 receptors and lose their “killing” ability. As a result, the body cannot effectively resist the infection or tumors, which can lead to the shutdown of the immune system. A current method to overcome this problem is by using checkpoint inhibitors, such as anti-PD-L1 or anti-PD-1 antibodies. Despite certain improvements in T cell responses, the major drawbacks of antibody therapy are its non-specificity, which leads to serious side-effects and autoimmune response, as well as its high cost. Therefore, finding a safer and more affordable solution would greatly benefit the field of immunomodulation and checkpoint inhibition. In this project, we propose a modular multicompartment trigger-responsive system that can potentially inhibit PD-L1 on the tumor and its exosomes. The system consists of multicompartment lipid vesicles, functionalized with NIR-responsive gold nanorods and magneto-responsive iron nanoparticles, which enable the rupture of the vesicle and its manipulation in space. Furthermore, the presence of tumor-recognition ligand on the vesicle is expected to provide the specificity for the selected tumor, while components such as CD47 and PEG would prolong the circulation time in the system and prevent unspecific interactions. Finally, selected immunomodulation agents could potentially facilitate the blockade of PD-L1 on the tumor cells and their exosomes. This modular vesicle, when injected in proximity to the tumor, could be moved to a location of interest through the magnetic field and then ruptured with the NIR light. The irradiation would cause the release of the immunomodulation components, which could restore the activity of the T cells and thereby recover the immune system.

Ashley Tafoya

Ashley N. Tafoya*, Aidira D.Y. Macias Gonzalez, Jacqueline De Lora, Nick J. Carroll, Gabriel P. López

poster number 14

Comparative study of methods for making cell-like compartments containing membrane-less organelles

Compartmentalization is an essential hallmark of living cells, allowing for control over distinct inter-cellular and intra-cellular biological functions. A major obstacle to the fabrication of synthetic cells is the ability to engineer membrane-bound and membrane-less synthetic organelles in a cell-mimicking environment. Similar to the proteins that comprise membrane-less organelles, elastin-like polypeptides (ELPs) contain regions of disorder, are stimulus-responsive proteins, and undergo liquid-liquid phase separation (LLPS), allowing them to function as components of synthetic membrane-less organelles. Finding a method for forming giant unilamellar vesicles (GUVs) that are mechanically and chemically stable enough to incorporate ELPs would allow for a simplified cell model to be studied. It would be a step closer to engineering a life-like structure from nonliving matter for the ultimate goal of better understanding natural cells. Our goal is specifically to load an ELP into a synthetic cell-like architecture to make synthetic membrane-less organelles. This work investigates several encapsulation techniques for this purpose. The three techniques studied here include: (1) one-pot production of droplet stabilized GUVs relying on a charge-mediated fusion of SUVs inside of a surfactant-stabilized droplet which can be released to an aqueous environment as a GUV, (2) glass capillary microfluidic production of monodisperse GUVs, and (3) formation of supported lipid bilayers on porous silica microparticles. The one-pot platform is a simple and straightforward method with capacity to be scaled up and to create complex GUV-based synthetic cells in physiologically relevant environments. The microfluidics platform utilizes a water-oil-water (W/O/W) based double emulsion, which leads to dewetting of the oil phase creating monodisperse GUVs. The porous silica particles provide support for the lipid bilayer while allowing for the encapsulation of biological components within the pores of the beads. This study investigates the flexibility of each method for incorporating proteins and nucleic acids, the ability to tune sizes for different compartments, complexity, and overall yield.

Aukse Gaizauskaite

Aukse Gaizauskaite*, Henrike Niederholtmeyer

poster number 15

Engineering Biomolecular Condensates in Cell-Free Systems

Several proteins, that have intrinsically disordered regions (IDRs) and high multivalency, possess the ability to self-associate into molecularly crowded liquid-like droplets, so called biomolecular condensates. In nature, living cells employ condensates as membranelles organelles for spatial organization, which allows effective separation of chemical reactions, concentration of molecules, and regulation of reaction kinetics. Cell-free transcription and translation (TX-TL) systems have emerged as alternatives to living cells in bioengineering, but they are still lacking ways to compartmentalize molecules. In our approach, microfluidic techniques and specifically designed microfluidic devices are applied to characterize and engineer protein-condensates in cell-free systems. Micrometer size environments in microfluidic chips with diffusive supply of TX-TL reagents ensure long-term continuous protein synthesis with the constant supply of fresh reaction components. Synthetic protein condensates, as programmable and dynamic molecular assemblies, can be useful for the construction of life-like systems from the bottom up, as well as for the creation of biochemically active compartments by programming the sequestration of different molecules or signalling networks.

Bert Van Herck

Bert Van Herck*, Gijsje Koenderink, Cees Dekker

poster number 16

Development of an integrated lab-on-a-chip system to establish an artificial life cycle of synthetic cells

Recent advances in synthetic biology considerably accelerated the progress toward developing synthetic cells. One crucial step in the process of generating an autonomous synthetic cell is the establishment of a cell cycle that mimics the sequence of events that takes place as the cell grows and divides. Many of these elements have already been individually recapitulated in vitro. Now the time has come to combine these modules into an integrated system of growing and dividing liposomes, mimicking a continuous life cycle of a living cell. For this purpose, we are developing an integrated lab-on-a-chip system that can be used as a fundamental tool to develop a living synthetic cell that eventually can undergo successive evolutionary cycles. More specifically, we will expand Octanol-assisted Liposome Assembly (OLA), for the production of cell-sized liposomes, and trap the liposomes in microfluidic structures for further manipulation according to our need to establish on-chip growth, division, and selection. We will present our first steps in developing the microfluidic device, and provide an outlook on how we plan to integrate these modules into one single microfluidic device.

Cathrine Abild Meyer

Abild Meyer, C.*, De Dios Andres, P., Brodszkij, E., Westensee, I. N., Lyons, J., Vaz, S. H., Städler, B

poster number 17

Crosslinked hybrid vesicles and their interactions with astrocytes cultured in paper-based chips

Astrocytes have been attracting increasing attention through the last couple of years due to their newly discovered role in neurodevelopment and function in diseases and under physiological conditions. However, the understanding of how astrocytes interact and respond remains scarce. Here, we explore how polymer lipid hybrid vesicles associate with the astrocytic cell line C8-D1A or primary cells cultured in polystyrene tissue culture or floating paper-based chips. The paper-based chips provided a 3D scaffold for the cells to mimic the physiological tissue. Consequently, they allowed the astrocytes to form a 3D network with preserved viability, calcium signaling ability, and glial fibrillary acidic protein expression. Hybrid vesicles consisting of the amphiphilic block copolymer poly(cholesteryl methacrylate)-block-poly(2-carboxyethyl acrylate) and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine lipids, which can be crosslinked with EDC/NHS chemistry, and offer a potential useful scaffold for artificial organelles. The hybrid vesicles show no short-term toxicity to the C8-D1A cell line or the primary astrocytes. Finally, the internalization of the hybrid vesicles by the astrocytes was confirmed when cultured in the paper-based chips. These findings are a step toward creating a more stable scaffold for artificial organelles and contribute to our fundamental understanding of the interaction between astrocytes and soft-material-based nanoparticles. Abild Meyer, C., De Dios Andres, P., Brodszkij, E., Westensee, I. N., Lyons, J., Vaz, S. H., Städler, B., Astrocytes in Paper Chips and Their Interaction with Hybrid Vesicles. Adv. Biology 2023, 2200209. https://doi.org/10.1002/adbi.202200209

Mengfei Gao

Mengfei Gao*, Dishi Wang, Weihua Leng, Michaela Wilsch-Bräuninger, Dietmar Appelhans, T-Y. Dora Tang

poster number 32

Cell free expression in native protein-PNIPAAm based proteinosome

Artificial cell systems have rapidly advanced through the use of cell free expression, allowing the constructions of predicable and programmable behaviours in vitro. But the current approaches are often limited by long term stability and resources that can access the interior of a membrane bound compartment. To address these challenges, we introduce a new method to produce protein-polymer protocells (proteinosomes) directly with native proteins that are stable, porous and can support cell free transcription and translation. Furthermore, we demonstrate the feasibility of regulating and compartmentalizing cell free expression reactions through tuning the protein-polymer crosslinking. Our simplified methodology provides a versatile route to establishing robust synthetic cells that provide architectural features close to a nuclear pore as well as offering a promising platform for developing sophisticated gene circuits by combining multiple proteinosome systems in cell free expression-based reactions

Paula de Dios Andres

Paula De Dios Andres*, Joseph Lyons, Brigitte Städler

poster number 36

Towards an Artificial Cell with Transmembrane Asymmetry

Bottom-up synthetic biology is an approach that uses designed building blocks to assemble life-like units. The membrane of living cells and their organelles have commonly transmembrane asymmetry, but their synthetic counterpart rarely have this property. The bottom-up assembly of polymer-containing vesicles typically results in random distribution of the membrane constituents. Polymer-lipid hybrid vesicles are an alternative to liposomes and polymersomes that combine the self-assembly ability of the lipids and benefits of modern polymer chemistry. Only limited reports show hybrid vesicles with transmembrane heterogeneity, obtained due to the assembly process. However, ideally, transmembrane asymmetry can be regulated post-assembly. Here, we explore the use of lipid flippases, which are reconstituted in polymer-lipid hybrid vesicles, to re-arrange the anionic lipids to obtain transmembrane asymmetry. We employed hybrid vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, 1,2-dioctanoyl-sn-glycero-3-phospho-(1'-myo-inositol-4'-phosphate) and the amphiphilic block copolymer poly(cholesteryl methacrylate)-block-poly(2-carboxyethyl acrylate) to reconstitute P4-ATPase Drs2p-Cdc50p flippase. The ATP depletion confirmed the presence of the flippase in the hybrid vesicle membrane. An Annexin-binding assay in small and giant hybrid vesicles was used to illustrate the flipping of phospholipids. Taken together, the transmembrane transport of phospholipids is the first step towards the control over the location of the membrane constituents in artificial cells with the potential for specifically tuning the lipid orientation mimicking the behaviour of mammalian cells.

Samuel Chen

Yen-Yu Hsu*, Samuel J. Chen*, Allen P. Liu

poster number 37

Building mechanically activated synthetic cells with calcium-induced exocytosis

Exocytosis is essential for cell-cell communication in multicellular organisms and is important for numerous biological functions. In cells, exocytosis requires membrane fusion mediated by SNARE proteins, whose activities are calcium dependent. Several non-native membrane fusion mechanisms have been demonstrated; however, few are capable of sensing and responding to environmental stimuli. Here, we aim to develop synthetic cells that couple mechanosensing to calcium-triggered exocytosis in a vesicle-in-vesicle system. Artificial exocytosis is mediated by first anchoring DNA oligonucleotides on opposing membranes. Membrane fusion is controlled by shielding DNA oligos on both surfaces with membrane-bound, calpain-cleavable PEG polymers. As calpain is calcium-activated, an influx of calcium into a giant unilamellar vesicle (GUV) causes cleavage of PEG polymers, enabling membrane fusion between the encapsulated small unilamellar vesicles (SUVs) with the GUV, thus releasing the SUV contents. Mechanosensitive capabilities are introduced by cell-free reconstitution of mechanosensitive channel of large conductance (MscL) on GUV membranes . Using membrane fusion and content mixing experiments, control over DNA oligo-mediated membrane fusion on SUV-GUV, SUV-SUV, and SUV-lipid-coated bead systems were demonstrated. Furthermore, functional reconstitution of MscL on GUVs was confirmed by observing activation of MscL via osmotic pressure and the subsequent calcium influx into GUV. This new system is the first to recapitulate the coupling of calcium influx to trigger secretion in a synthetic cell model, which mimics the biochemical activation of natural SNARE-mediated membrane fusion machineries. This platform could see applications in smart drug delivery, as well as uses in exploring communications between synthetic and natural cells.

Yen-Yu Hsu

Yen-Yu Hsu*, Samuel Chen*, Allen Liu

poster number 44

Building mechanically activated synthetic cells with calcium-induced exocytosis

Exocytosis is essential for cell-cell communication in multicellular organisms and is important for numerous biological functions. In cells, exocytosis requires membrane fusion mediated by SNARE proteins, whose activities are calcium dependent. Several non-native membrane fusion mechanisms have been demonstrated; however, few are capable of sensing and responding to environmental stimuli. Here, we aim to develop synthetic cells that couple mechanosensing to calcium-triggered exocytosis in a vesicle-in-vesicle system. Artificial exocytosis is mediated by first anchoring DNA oligonucleotides on opposing membranes. Membrane fusion is controlled by shielding DNA oligos on both surfaces with membrane-bound, calpain-cleavable PEG polymers. As calpain is calcium-activated, an influx of calcium into a giant unilamellar vesicle (GUV) causes cleavage of PEG polymers, enabling membrane fusion between the encapsulated small unilamellar vesicles (SUVs) with the GUV, thus releasing the SUV contents. Mechanosensitive capabilities are introduced by cell-free reconstitution of mechanosensitive channel of large conductance (MscL) on GUV membranes. Using membrane fusion and content mixing experiments, control over DNA oligo-mediated membrane fusion on SUV-GUV, SUV-SUV, and SUV-lipid-coated bead systems were demonstrated. Furthermore, functional reconstitution of MscL on GUVs was confirmed by observing activation of MscL via osmotic pressure and the subsequent calcium influx into GUV. This new system is the first to recapitulate the coupling of calcium influx to trigger secretion in a synthetic cell model, which mimics the biochemical activation of natural SNARE-mediated membrane fusion machineries. This platform could see applications in smart drug delivery, as well as uses in exploring communications between synthetic and natural cells.

Emma Gehlbach

Emma Gehlbach*, Abbey Robinson, Katarzyna Adamala

poster number 51

Using sequential encapsulation to improve encapsulation efficiency of liposomes formed by gentle hydration

Synthetic cells are simple, man-made objects that mimic the structure and function of living cells and can be programmed to produce proteins through cell-free protein synthesis (CFPS). Liposomes are spherical lipid bilayers made of amphiphilic lipids that can be used to form synthetic cells by mimicking the structure and function of the plasma membrane. Liposomes can be created through several methods, including gentle hydration of lipids with aqueous buffer. Gentle hydration has an advantage over other some other methods of liposome formation because it makes more pure liposomes without organic contaminants in the lipid bilayer. However, compared to other methods of liposome formation, gentle hydration is less efficient at encapsulating aqueous solution within the liposome, which is a necessary step for making synthetic cells that can perform CFPS. Improving the encapsulation efficiency of the gentle hydration process would make it a more useful technique for synthetic cell research. We hypothesized that adding lipids to the buffer solution in increments (sequential encapsulation) instead of all at once (non-sequential encapsulation) would increase the encapsulation efficiency of gentle hydration. We tested this method in HEPES buffer (used in CFPS) and CHAN buffer (used for liposome storage) using cholesterol and POPC lipids, as they mimic mammalian membrane lipid composition. We used lipid concentrations ranging from 1-9 mM. We found that sequential encapsulation increases percent encapsulation of liposomes in HEPES buffer by up to 3.5-fold, but sequential encapsulation does not increase percent encapsulation of liposomes in CHAN buffer.

Session Applications: anything useful and moneymaking, like medicine, bioengineering etc. posters

Aidira D. Y. Macias Gonzalez

Aidira D. Y. Macias Gonzalez*, Jacqueline De Lora, Ashley N. Tafoya, Matthew L. Lakin, Joachim P. Spatz, and Gabriel P. Lopez

poster number 11

Developing Biosensing Coatings by Embedding SynCells in Porous Silica Films

Porous silica coatings hold promise for biomedical and sensor applications due to their biocompatibility and flexible structural and chemical attributes. We describe the fabrication of hybrid materials using biofriendly sol-gel chemistry for biomedical applications. Our long-term goal is to develop porous silica coatings that incorporate synthetic cells (SynCells) that can sense and respond to exogenous biochemical cues. Giant unilamellar vesicle (GUV)-based SynCells entrapped in porous silica films might offer this possibility. To produce porous materials, we use tetramethyl orthosilicate as a precursor, with a low molecular weight polyethylene glycol to conserve the integrity of rudimentary models for SynCells entrapped within the silica film. The sol-gel chemistry employed differs from conventional methods as it does not use methanol or other alcohols as a cosolvent and potentially harmful catalysts and byproducts. Vaporization is used to eliminate the methanol formed during silica condensation in the sol-gel process. We then entrap SynCells, comprising GUVs, to preserve their stability for long periods of time and show that this further depends on storage temperature of either 20 or 4°C. SynCells entrapped in silica matrices exhibit long-term stability especially when stored at lower temperature. We present easy to fabricate hybrid, porous silica matrices that extend the structural integrity of embedded GUV-based SynCells and probe chemical communication using an aptamer-steroid sensor platform to determine the ability to interact with small molecules that can freely diffuse through the porous silica matrix.

Ana Maria Restrepo

Ana María Restrepo*, Zhanar Abil, Christophe Danelon.

poster number 12

CADGE: Clonal amplification-enhanced gene expression for cell-free directed evolution

In cell-free gene expression, low input DNA concentration severely limits the phenotypic output, which may impair in vitro protein evolution efforts. We address this challenge by developing CADGE, a strategy that is based on clonal isothermal amplification of a linear gene-encoding dsDNA template by the minimal Φ29 replication machinery and in situ transcription-translation. We demonstrate the utility of CADGE in bulk and in clonal liposome microcompartments to boost up the phenotypic output of soluble and membrane-associated proteins, as well as to facilitate the recovery of encapsulated DNA. Moreover, we report that CADGE enables the enrichment of a DNA variant from a mock gene library either via a positive feedback loop-based selection or high-throughput screening. This new biological tool can be implemented for cell-free protein engineering and the construction of a synthetic cell.

Hannah Sleath

Hannah Sleath*, Janna Lowensohn, Bortolo Mognetti, Yuval Elani, Lorenzo Di Michele

poster number 21

Engineering Artifical Cell Chemotaxis using DNA Nanotechnology

One key goal of the field of nanomedicine and drug delivery is to achieve long-range targeting of diseased tissues, to simultaneously increase the effectiveness and decrease the toxicity of therapeutic treatments. Diseased tissues often release substances as signals to the immune system, such as in the inflammatory response. Therefore, one potential solution for targeted drug delivery is to design artificial cells that can move up a concentration gradient of the signalling substance to its source, a type of motion known as chemotaxis. The aim of this work is to engineer chemotactic artificial cells and use them to study the multivalent interactions between molecular “receptors” embedded in the cell membranes and “ligands” in the surrounding environment. We have established an experimental system in which DNA-functionalised giant unilamellar vesicles (GUVs) perform chemotactic motion by migrating along a surface density gradient of ligands adsorbed to a surface. A variety of system parameters and their effects on the motion have been explored, including varying the binding and unbinding rates of the ligand-receptor pairs, varying the surface density of DNA constructs, and varying the size of the GUVs. Experimental outcomes are compared with stochastic computer simulations, to investigate the theory governing the motion of the GUVs over ligand-coated surfaces and the effect that the ligand gradients have on this motion. The current focus of the work is to optimise an updated version of the experimental system, in which a catalytic mechanism involving toehold-mediated strand displacement is employed to boost the speed of GUV motion.

Judee Sharon

Judee A. Sharon*, Chelsea Dasrath, Aiden Fujiwara, Alessandro Synder, Mace Blank, Sam O’Brien, Lauren M. Aufdembrink, Aaron E. Engelhart, Katarzyna P. Adamala

poster number 23

Trumpet: a new operating system for biocomputing

Biological computation is becoming a viable and fast-growing alternative to traditional electronic computing. Here we present a new biocomputing technology called Trumpet: Transcriptional RNA Universal Multi-Purpose GatE PlaTform. Trumpet combines the simplicity and robustness of the simplest in vitro biocomputing methods, adding signal amplification and programmability, while avoiding common shortcomings of live cell-based biocomputing solutions. We have demonstrated the use of Trumpet to build all universal Boolean logic gates. We have also built a web-based platform for designing Trumpet gates and created a primitive processor by networking several gates as a proof-of-principle for future development. The Trumpet offers a new paradigm in biocomputing, providing an efficient and easily programmable biological logic gate operating system.

Kaitlin Eversole

Kaitlin Eversole*, Peter Davenport, Matthew R. Lakin

poster number 25

Programmable genetic networks for synthetic cell engineering

Synthetic cells are a promising approach for advanced therapeutics, bioproduction, and diagnostics. However, the genetic devices that control cellular phenotype and behavior are often hardwired and cannot be modified inside the host cell, thereby constraining design space and current applications. To address this limitation, we engineered genetic constructs capable of switching between multiple configurations in situ, thus enabling programmable control of cellular behavior. This poster will present preliminary results from the development and optimization of recombinase-programmable genetic circuits, capable of being primed for subsequent molecular computations through site-specific enzymatic recombination. To simplify the rewiring process and expand recombinase-programmable circuit design space, CRISPR transcription factors were utilized, thus facilitating straightforward scalability for future multilayer designs. Similar bottom-up engineering approaches could be used in future work to build genetic circuits capable of sensing and autonomously reprogramming the behavior of rationally engineered synthetic cells as determined by previous experiences.

Marco Oliveira

Marco Oliveira*, Rayane Lima, Lilian Hasegawa, Mariana Severo, Leila Barros, Cristiano Lacorte, Elibio Rech

poster number 27

Int-Plex@ (INTegrase PLant EXpression): a serine-integrase-based genetic switch system for inversion and excision of target genes inserted in the genome of Nicotiana benthamiana

Serine-integrases are a promising biotechnological tool to modulate plant metabolic pathways and regulate gene expression under controlled conditions. Initially present in nature as a mechanism for phage DNA integration into bacterial host genomes, these recombinases can rearrange long sequences of DNA in different ways, as excision or sequential inverting events turning on and off a target gene expression in a reversible and specific manner, according to the manipulation of their recombination sites pairs’ sequences, positions and distribution on a synthetic construct. In this work, we showed the assembly and operation of Int-Plex@, a genetic switch system based on applying four distinct prophage-derived serine-integrases as an input trigger mechanism for the inversion or excision of a target gene stably integrated into the genome of Nicotiana benthamiana. The genetic switch was formed by a mGFP gene sequence flanked by a combination of attachment sites for serine-integrases Bxb1, PhiC31, Int 13 and Int9. It can be divided into two modules: an inversion and an excision modules. The transient expression of Int9 or Int13 activates the inversion module. In this module, the activation results in the mgfp sequence being flipped to its forward orientation with the resulting expression of the mGFP reporter as a switch output. The excision module is activated by transient expression of Bxb1 or phiC31. In this case, the DNA sequence flanked by their attachment sites is irreversibly excised from the genome. The inputs can be activated sequentially, turning the target expression on and off or deleting the cassette thoroughly after modulations. Different delivery strategies for serine-integrase heterologous terms were successfully tested, although yielding varying levels of activation efficiency: leaf tissue agroinfiltration of agrobacterium transformed with viral vector Potato virus X (PVX) or with binary vector pCAMBIA2300 and biolistic system. This memory switch system can be used in future genetic circuits to engineer and modulate plant metabolic pathways of economic and environmental importance while also addressing transgene biocontainment issues, given the possibility of cassette excision.

Meline Macher

Meline Macher*, Rahul Rimal, Assa Yeroslaviz, Rinho Kim, Saradaprasan Muduli, Eva Bertosin, Smriti Singh, Ilia Platzman, Joachim P. Spatz

poster number 31

Bottom-up Assembly of Fully Synthetic Extracellular Vesicles against Inflammatory Skin Disease

Extracellular Vesicles (EVs) are naturally produced compartments for intercellular communication. Depending on their biochemical composition, they have intrinsic therapeutic potential against various physical impairments such as cardiovascular, immune and skin diseases. Especially the pro-regenerative and immunomodulatory effects of human mesenchymal stem cell (hMSC)-derived EVs are in the focus of biomedical research and technology. Despite the large biomedical potential of EVs, their conventional enrichment from cell culture medium hampers translation into clinics, as natural EV biogenesis is inherently difficult to control. This leads to high EV heterogeneity and challenges in production and mechanistic understanding. To overcome these limitations, we developed and apply bottom-up synthetic biology for a quantitative and well-controlled production of liposome-based fully synthetic EVs (synEVs). The goal of our research is to specifically mimic adipose-derived hMSC EVs and to achieve similar functionalities using synEVs in a human organotypic tissue model of inflammatory skin disease in vitro. To this end, we have isolated hMSC EVs and characterized their biophysical and biochemical properties. To cover the size range of natural EVs, we have produced two types of synEVs, based on either small (diameter below 200 nm) or large (200-1000 nm) unilamellar vesicles, both with a lipid composition and a slightly negative surface charge mimicking natural EVs. We have identified abundant surface proteins and miRNAs in the hASC EVs and have been testing the corresponding synthetic miRNAs and recombinant protein domains as active cargo of the synEVs. We have found that after topical application onto skin models, synEVs penetrate through the epidermis and even into the dermal layers. We show that a set of 5 surface proteins on synEVs is sufficient to improve histology and restore epidermal thickness and differentiation in the inflamed skin models. This is particularly interesting as it demonstrates direct effects of synEVs on skin cells without immune cell contributions. To identify the molecular mechanisms of synEVs, we are now analysing our transcriptomics data from the healthy, diseased and synEV-treated tissue models. Further optimization of synEV composition will be required to dissect the function of individual EV components. The developed synEVs have the potential to overcome challenges in the clinical translation of EVs and present a potential novel therapeutic for inflammatory skin diseases as well as a tool to better understand the mechanisms of action of natural EVs.

Sung-Won Hwang

Sung-Won Hwang, Hossein Moghimianavval, Cong Truc Huynh, Chung-Man Lim, Eben Alsberg, Nicholas A. Kotov, Allen P. Liu

poster number 40

Mechano-responsive self-degradable hydrogel using giant unilamellar vesicles

Scaffold-based tissue engineering requires the scaffold to degrade as cells proliferate and a new extracellular matrix develops. However, it is difficult to predict tissue regeneration profile and to engineer a scaffold that degrades by itself. Given that tissue growth generates pressure, we have developed a hydrogel that can degrade in response to compressive stress. To introduce mechanoresponsive behavior to a calcium-crosslinked alginate hydrogel, we employed giant unilamellar vesicles (GUVs) loaded with a gel-degrading compound, ethylene glycol tetraacetic acid (EGTA). EGTA-loaded GUVs were generated using a continuous droplet interface crossing encapsulation technique, mixed with alginate, and stiffened with calcium chloride solution. The embedded GUVs in the gel were found to be stable over 2 weeks. When the gel was subjected to compressive stress, the GUVs in the gel matrix ruptured and released EGTA, causing the hydrogel to degrade. We characterized a gel-to-solution transition by analyzing the Brownian motion of fluorescent beads embedded within the gel. Stiffness and the residual gel amount after applying pressure were also analyzed and have shown significant reduction. The results demonstrate that the GUV-embedded hydrogel can be a promising material for various biomedical applications, including tissue engineering and drug delivery, that require rapid and on-demand degradation of drug release in response to compression.

Sparky Mitra

Sparky Mitra*, Yanzhe Liu*, Andrew Nguyen*, Aniket Dhar*, Amit Klein*, Wesam Kanim*

poster number 46

Carbon Fixation in a Minimal Cell

Atmospheric carbon dioxide release has been increasing, and resulted in changes in global temperature. Uncontrolled release of greenhouse gasses brings an urgent and existential threat for organisms on Earth. Current carbon capture technologies aim to use industrial processes to convert carbon dioxide into other forms, or mitigate their release into the atmosphere through renewable energy forms. Our team proposes the use of synthetic biology to create an artificial organism able to capture carbon more efficiently, leveraging natural and synthetic carbon fixation pathways. One such pathway, the POAP cycle, has fewer reactions than any natural or synthetic pathway to date, totalling only four reactions. This pathway will then be inserted into the J. Craig Venter Institute’s Syn 3.0 minimal cell that has been evolved using TALE from 37°C to 43.4°C (via growing cells at previous bottleneck temperatures) to create a minimized organism that can perform thermodynamically-feasible carbon sequestration in isolation. The genes are inserted into Escherichia coli for assembly with two being unequivocally successful. So far, evolution and pathway assembly have been successful with other steps moving forward as well boding well for ultimately successful transformation of the minimal cell. The transformed E. coli is then purified, after which evolved minimal cells are transformed with assembled constructs. Then oxalate production is then measured by LC/GC-MS to assess initial efficiency of the synthetic pathway in vivo. For optimal efficiency, the genomes of the engineered cells will eventually be transplanted with genes assisting ferredoxin protection that will directly aid the POAP cycle. In the future, the efficiency of the organism can be measured through carbon-14 labeling within the cells to compare against other known rates. The transformed cells can also be tested in conjunction with bioreactors to assess if efficiency can be increased via environmental stimuli for further applicability to large-scale carbon sequestration.

Session Building blocks: metabolism, gene replication, membrane synthesis, ribosome assembly etc. posters

Gaku Sato

Gaku Sato, Nobuhide Doi, Kei Fujiwara

poster number 20

In vitro reconstitution of glycolysis as the core metabolism of synthetic cell

Glycolysis is a central metabolic system found in almost all living cells, producing energy and precursors for important biomolecules. However, the complexity of glycolysis has hindered its application in synthetic cell research. In this study, we developed a kinetic model to overcome the complex behavior of glycolysis and applied it to the energy regeneration system of the PURE system, an in vitro protein synthesis system using purified elements. We then attempted to synthesize important biomolecules from glucose with high yields and found that 100% conversion of glucose can be achieved by adding ATPase. Theoretically, any ATP-consuming system, including protein synthesis and genome replication, could replace ATPase. Our findings contribute to the construction of a synthetic cell with glycolysis as its core metabolic system.

Joseph Heili

Joseph Heili

poster number 22

Activation of caged functional RNAs via oxidative desulfurization of 2-thiouridine

The origins of life field, or abiogenesis, seeks to understand the processes by which life arose from non-living matter. My favorite area of research in this field is the RNA world hypothesis, purporting that RNA preceded both DNA and proteins evolutionarily. This hypothesis relies on initial abiotic generation of nucleosides and simple RNA polymers and researchers have demonstrated templated nonenzymatic polymerization of RNA rich in GC sequences using activated nucleotide derivatives. In contrast, AU-rich sequences exhibit diminished fidelity and speed. This is due in part to G-U wobble pairing, which is ca. isoenergetic with A-U base pairing. Recently, it was shown that substitution of 2-thiouridine (2sU) for U resulted in diminished wobble pairing and stronger base pairing with A, enabling faster, higher-fidelity base pairing. While this solves the problem of wobble pairing in nonenzymatic RNA copying, it brings about a second issue. In functional RNA, which are critical to the RNA world hypothesis, G-U wobble pairs are common secondary structure elements and metal ion binding motifs. Taking advantage of a recent finding that 2sU can be oxidatively desulfurized to U, we have explored the potential for rescue of functionally caged RNA polymers initially transcribed with 2sU. In experiments with a fluorescent aptamer and a ribozyme nuclease, each reliant on critical interactions with U for proper folding, we have shown that the 2sU RNA exhibiting diminished function can be at least partially rescued via the oxidative desulfurization reaction. Because the oxidative desulfurization reaction is prebiotically plausible these findings support the hypothesis that RNA polymers could be replicated non-enzymatically while also evolving function. Looking forward, I am characterizing the kinetics of the oxidative desulfurization reaction and identifying further functional RNA targets that rely critically on U for proper folding.

Kai Kean

N/A

poster number 24

N/A

N/A

Marijn van den Brink

Marijn van den Brink

poster number 29

Evolutionary optimization of synthetic genomes by image-assisted selection of liposomes

Synthetic cells provide a minimal model system to study fundamental principles of life. One approach to the construction of synthetic cells requires the stepwise integration of primary cellular functions, such as DNA replication, cell growth and division. We aim at accelerating the optimization and integration of these biological functions in gene-expressing liposomes using laboratory evolution. One essential step is the selection of liposomes displaying the desired phenotype from a large population of genetic variants. We are developing a microscopy-based selection method enabling high-resolution image acquisition for phenotypic interrogation and real-time labelling of synthetic cells via photoactivatable fluorophores, whereafter the selected vesicles are sorted by fluorescence-activated cell sorting. This technique has the potential for high-throughput selection of liposomes based on multidimensional features, including vesicle shape, protein localization and dynamic behaviours, which is indispensable for directed evolution of complex synthetic genomes. We show that liposomes can be selectively labelled using a confocal microscope and subsequently isolated from the liposome pool. By integrating artificial intelligence-assisted detection of the ‘fittest’ liposomes, we aim to develop an automated platform for rapid identification and sorting of synthetic cells.

Mireia Casanovas Montasell

Mireia Casanovas Montasell*, Alexander N. Zelikin

poster number 33

Chemical zymogens: synthetic proenzymes based on the protein cysteinome

In nature enzymes are activated and deactivated on demand, forming the basis of signal transduction while maintaining enzymatic homeostasis. These interconversions between an active enzyme to its inactive form are performed through different mechanisms, and are highly specific in space and time. However, synthesis of zymogens using tools of organic chemistry and chemical biology remains a big challenge. In this work, we address this task and engineer chemical zymogens based on the protein cysteinome. We use the protein cysteine groups to create several types of synthetic zymogens, all based on reversible conjugation of the cysteine thiol to a blocking group. Through this, we obtain inactive enzymes, which then we can reactivate on demand. In one design, we engineer enzyme (de)activation via artificial protein-protein interactions. In another, we take steps towards establishing specificity of enzyme recovery. In yet another example, we engineer a system whereby enzyme activity is controlled by steroids, in a manner that mimics nature. The specific application for the developed platform is the design of artificial signalling cascades in fully synthetic cells, and we believe it represents a big step forward in the field of synthetic, chemical zymogens.

Miyer Patiño-Ruiz

Miyer Patiño-Ruiz*, Bauke F. Gaastra, Zaid Ramdhan Anshari, Dirk J. Slotboom, Bert Poolman

poster number 34

Providing energy for the uptake of building blocks in a synthetic cell

The cell membrane constitutes a protective barrier that keeps a defined and life-friendly interior separated from an external and sometimes hostile environment. Rather than being a static physical barrier the membrane is a dynamic structure constituted mostly by lipids and proteins. Indeed, the functional activity of the cell membrane is intimately associated with the proteins embedded in this hydrophobic fluidic mosaic. Integral membrane transporters are essential to keep the cell homeostasis by mediating the uptake of nutrients and at the same time discarding waste products whose accumulation might be deleterious for life. Although a fraction of these transport processes can occur via diffusion of small molecules (passive or facilitated) it is well known that the cell invests a considerable portion of the energy budget in the uptake of nutrients and export of waste metabolic products [1], which are in most cases charged or large hydrophilic molecules. We have recognized this active transport as an essential and desirable feature for any potential synthetic cell designed to mimic the of out-of-equilibrium conditions of actual living cells [2]. We have previously demonstrated that the arginine degradation pathway provides sufficient energy in the form of ATP to activate the uptake of an osmolyte in response to an osmotic shock [3] and even to eventually energize the synthesis of lipids [4]. In this work, we present a simple strategy to provide a synthetic cell with free energy in the form of a proton electrochemical gradient that can be used to drive the translocation of building blocks across the lipid membrane. For that, we use a precursor-product system existing in fermentative bacteria as secondary source of proton motive force based on the decarboxylation of L-malate [5]. We reconstituted the L-malate decarboxylation pathway from Lactococcus lactis in lipid vesicles and demonstrated the sustained generation of a chemical proton gradient and an electrical gradient over a period of several hours. By co-reconstitution of the Escherichia coli H+ symporters GltP and LacY we showed that the free energy from the L-malate decarboxylation stored as proton motive force can drive the sustained accumulation of glutamate and lactose at concentrations compatible with those required for competitive metabolism in living cells. [1] R. Milo, R., Phillips, Cell Biology by the Numbers (Garland, Science 2016). [2] Sikkema, H. R.; Gaastra, B. F.; Pols, T.; Poolman, B. Cell Fuelling and Metabolic Energy Conservation in Synthetic Cells. ChemBioChem. 2019, pp 2581–2592. [3] Pols, T.; Sikkema, H. R.; Gaastra, B. F.; Frallicciardi, J.; Śmigiel, W. M.; Singh, S.; Poolman, B. A Synthetic Metabolic Network for Physicochemical Homeostasis. Nat Commun 2019, 10 (1) [4] Bailoni, E.; Poolman, B. ATP Recycling Fuels Sustainable Glycerol 3-Phosphate Formation in Synthetic Cells Fed by Dynamic Dialysis. ACS Synth Biol 2022, acssynbio.2c00075. [5] Poolman, B., Molenaar, D., Smid, E., Ubbink, T., Abee, T., Renault, P. P., Konings, W. N., Malolactic fermentation: electrogenic malate uptake and malate/lactate antiport generate metabolic energy. J. Bacteriol. 1991. 19, 6030-6037.

Shanny Ackerman

Shanny Ackerman*, Patricia Mora-Riamundo, Avi schroeder

poster number 38

Energy pathways in synthetic cells

The success of synthetic cells (SCs) in many translational applications requires their prolonged and dynamic activity, which heavily relies on energy availability. Without an external energy supply, the maximum protein production capacity inside SCs was measured in a few hours. Different techniques have shown successful production of ATP inside SCs. However, most methods were based on structural changes and the addition of complex enzymatic reactions to the SCs. Nevertheless, finding a simple solution to the energy supply challenge is essential for advancing applications with SCs, especially medical ones. This study aims to introduce a human-made and uniquely structured energy-supplying organelle into the SCs and prolong their protein production for several days.

Tanner Hoog

Tanner Hoog*, Matt Pawlak, Nathan Gaut, Katarzyna Adamala, and Aaron Engelhart

poster number 42

Utility of the Selective Denaturant Perchlorate in Origins of Life and Synthetic Cell Research

The Hofmeister ions are categorized by their ability to increase biomolecular secondary structure stability (kosmotropes) or to decrease biomolecular secondary structure stability (chaotropes). Here we show that the G quadruplex, a noncanonical nucleic acid secondary structure, retains stability in solutions of anionic chaotropes relative to several other nucleic acid secondary structures, including a duplex and i-Motif. This relative insensitivity of G quadruplexes compared to duplexes enables a switchable nucleic acid electron transfer device, which can be switched using heat or dilution. Additionally, we show that ribozymes retain activity in perchlorate brines. This is of interest from an origins of life perspective because transient pools of liquid water can be stabilized on Mars due to perchlorate’s innate hygroscopicity.

Viktoriia Belousova

Viktoriia Belousova, Kareem Al Nahas, Petra Schwille

poster number 43

Mutation of alpha-hemolysin creates a cation-controlled transmembrane pore for synthetic cells

Transport of molecules across the membrane is crucial for cell communication, organization, and regulation. In nature, there are many sorts of membrane transporters, ion channels and pore proteins that are regulated and coordinated by complex signaling cascades. In synthetic cells based on biomimetic membrane compartments, implementing and regulating transport across the membrane remains a challenge. Ion channels and pores were reconstituted in synthetic cell mimics like GUVs before, but are often suboptimal modules to be regulated under the conditions of minimal systems. Generally, pore proteins that can be regulated by ions or small molecules constitute a more promising tool for this goal. In this work, we use a variant of Staphylococcus aureus pore protein alpha-hemolysin with a 5 amino acid mutation that makes it susceptible to Zn2+ ions. We demonstrate that this alpha-hemolysin mutant can be expressed in GUVs using a cell-free protein expression system and that it allows small molecules to diffuse across the GUV membrane. We also show that this passive transport can be controlled by addition of Zn2+ ions that bind to the pore protein and close it, blocking transmembrane diffusion. This variant of alpha-hemolysin thus appears to be a promising tool for future applications in synthetic cells that require the control of signaling and communication.

Ryota Miyachi

*Ryota Miyachi, Yoshihiro Shimizu, Norikazu Ichihashi

poster number 48

Transfer RNA synthesis-coupled translation and DNA replication in a reconstituted transcription/translation system

Cell-free or in vitro synthetic biology aims to reconstitute the biological functions from defined molecules, such as DNA, RNA, and proteins, to reveal the design principles of living systems and also provide new biotechnology (Wang et al., 2021). Despite various cellular functions have been reconstructed, however, the construction of an artificial self-reproductive system, which requires regeneration of the gene expression system from the gene expression system itself, remains a significant challenge. Especially, In vitro synthesis of Transfer RNAs (tRNAs), central factors in translation, and their coupled translation are important challenges in the construction of a self-regenerative molecular system (Hibi et al., 2020). Here, we first examined luciferase translation coupled with tRNA expression in a one-pot reaction. We tested different strategies for expression schemes with 5'-G and non-5'-G tRNAs and demonstrated expression of the 21 tRNAs that are minimally required for translation. Moreover, we showed DNA replication through expression of a tRNA encoded by DNA, mimicking information processing within the cell. Our findings highlight the feasibility of an in vitro self-reproductive system in which tRNAs can be synthesized from replicating DNA.

Evan Kalb

Mace Blank, Joshua Davisson, Evan Kalb, Angelin Prasad, Aaron Engelhart, Kate Adamala

poster number 52

Eutectic phase flexizyme reactions improve acylation and cell-free translation yield

Flexizymes are ubiquitous for acylating an enormous chemical diversity of compounds onto tRNA, enabling the translation of radically different peptide backbones, bonds, and side chains. However, flexizyme reactions suffer from several drawbacks such as single turnover kinetics, high magnesium carryover in in vitro translation reactions, and rapid hydrolysis. Utilizing a water ice-eutectic phase, we have developed novel flexizyme reaction conditions which demonstrate increased aminoacylation yields, low magnesium concentrations, and slow hydrolysis of the aminoacyl bond. Eutectic flexizyme reactions attain similar or higher yields than literature and maintain this high yield for 48+ hours, much longer than a standard flexizyme reaction. Slow hydrolysis means flexible flexizyme reaction timing and increased utility and flexibility of aminoacylation reactions. Furthermore, flexizyme reactions require an excess of MgCl2 (up to 600 mM). This concentration of Mg2+ has been shown to carryover into cell free protein synthesis reactions, inhibiting protein synthesis. Eutectic flexizyme reactions use 30 times lower MgCl2, reducing carryover and increasing peptide/protein production for in vitro translation reactions. Flexizyme reactions have been shown to be practically single turnover. Surprisingly, eutectic flexizyme reactions have increased turnover, further increasing aminoacylation yield. Overall, we present a new methodology for performing in vitro aminoacylation reactions, with increased yields and more flexible timing.

Scott Hansen

Grace Waddell, Benjamin Duewell, Naomi Wilson, Emma Drew, Sophia Doerr, Sarah Peabody, Andres Olavarrieta, Katie Faris, Reed Talus, & Scott D. Hansen*

poster number 53

Mechanisms controlling spatial patterning of PIP lipids in cell signaling

Cells interpret chemical cues in their environment and make decisions that control their fate. For example, human neutrophils respond to a variety of pathogenic signals by sensing, polarizing, and targeting the foreign pathogens. Critical for these cellular processes is the relay of signals between cell surface receptors, small GTPases, and phosphatidylinositol phosphate (PIP) lipids. Collectively, these molecules orchestrate numerous biochemical reactions at the plasma membrane that give rise to emergent properties, such as symmetry breaking, polarization, and spatial patterning during cell signaling. My lab uses a combination of biochemistry and quantitative cell biology to define the mechanisms that control these properties. Using supported membrane technology and single molecule imaging techniques, we have characterized several classes of lipid kinases and phosphatases that regulate the rapid amplification and down-regulation of PIP lipids in various spatial patterning programs. Long-term, we want to understand the design principles that allow PIP lipids to organize signaling reactions on cellular membranes. Given the critical role that PIP lipids play in cellular organization and signal transduction, our research will also help decipher mechanisms of misregulation that are associated with numerous human diseases.

Session Beyond the bench: education, outreach, ethics, biosafety posters

Orion Venero

Orion M Venero, Katarzyna P Adamala

poster number 35

Synthetic Systematics: The Question of Naming Synthetic Life

To fully apply the findings of synthetic cell research to a broader range of fields and research interests, it is paramount that there is a common language with which to discuss synthetic biotic constructs. In this respect, such systematization must be compatible with existing classification methodologies for terrestrial life: acting as an aesthetic or conceptual overlay atop existing systems of nomenclature. The development of a universal system of nomenclature for biology is deeply linked with the formulation of a universal theory of life because cogent systematization is contingent on semantically understanding the same logical and aesthetic problems faced when trying to proffer a universal theory of life. Evincing a universal theory of life is outside the scope of this effort, but the development of an aesthetic model which relates the classifiers ‘biotic’ and ‘living’ will allow for the formulation of universal systematics and perhaps aid future efforts to advance a universal theory of life. Accordingly, the biotic interval model, that we present here, demonstrates the precedent for, and feasibility of, the phylogenetic classification of synthetic life alongside classical terrestrial life.

William L Gannon (Bill)

William L. Gannon*, Department of Biology, Philosophy, and Graduate Studies, University of New Mexico, Albuquerque, NM 87131 (wgannon@unm.edu ) and, Gabriel P. Lopez, Department of Chemical and Biological Engineering, University of New Mexico, Albuquerque, NM 87131 (gplopez@unm.edu)

poster number 49

Increasing Accessibility of Emerging Technologies to URM Students: Lessons from a Graduate Student Seminar in Synthetic Cell Biology

Abstract -- The purpose of this research is to describe characteristics and motivations of students who have entered the university as undergraduate and graduate students to pursue research in synthetic cell biology, a field focused on engineering an artificial cell, synthetic cell or minimal cell as an engineered particle that mimics one or many functions of a biological cell. We provide details of a seminar course that was specifically created for students interested in synthetic cell biology and engineering with emphasis on both technological content and ethical considerations as this field emerges. Student participation included discussion, research, and presentation of student-identified projects. Given the special nature of the University of New Mexico as a minority-majority, Hispanic Serving Institution, the project is aimed to characterize the stories of students in the field about how they decided to enter a graduate program, what sort of barriers or hardships that they have encountered, where they hope their careers will develop, and what they feel about the future of synthetic cell research and applications. We describe how our synthetic cell biology course was conducted and provide recommendations for better teaching practices for future course offerings at other institutions derived from student feedback. We consider courses such as these to be effective ways to recruit and support current and potential students from underrepresented groups into the field.

Joshua Davisson

Joshua Davisson, Evan Kalb, Mace Blank, Kate Adamala

poster number 56

Complete genetic code reprogramming subverts current DNA purchasing biosafety measures

Highly conserved across all known life forms, the genetic code carries information transported by mRNA and translated into protein. Genetic code reprogramming alters this biological Rosetta Stone, liberating codons for the translation of noncanonical amino acids or to reassign a canonical amino acid into a new codon, weakening the link between nucleic acid and protein sequence. Recently, aminoacyl tRNA synthetase (aaRS)-free translation of proteins was demonstrated by supplementing aminoacylated tRNA into a protein synthesis using recombinant elements (PURE) reaction (Chen et al., 2021). This was accomplished using flexizymes, small synthetic ribozymes that charge tRNAs in vitro. However, flexizyme aminoacylation is not specific for any tRNA and can attach almost any amino acid to any tRNA. By aminoacylating all 20 amino acids to their noncognate tRNAs, complete genetic code reprogramming is possible, translating proteins using a novel genetic code. This genetically reprogrammed PURE reaction would enable the subversion of DNA purchasing biosecurity measures which heavily rely on sequence homology (Elworth et al., 2020). This is particularly relevant after a paper synthesized infectious poliovirus particles in vitro from assembled DNA oligos using a cell free protein synthesis reaction (Cello et al., 2002). The increasingly weak link between nucleotide and protein sequence highlights the need for further genetic biosafety considerations. This project demonstrates that genetic biosecurity and biosafety must adapt with novel genetic engineering tools like genetic code reprogramming.

Session Cell-free translation systems: the cytoplasm of synthetic cells posters

Matas Deveikis

Matas Deveikis

poster number 30

Exploring limiting factors in the PURE cell-free system

Cell-free protein synthesis (CFPS) systems have emerged as a valuable tool for protein production outside a living chassis. Protein synthesis using recombinant elements (PURE) cell-free system has been especially promising for use in engineering synthetic cells due to the ability to finely model the system and adjust the levels of its' components. However, comparatively low yields have made the PURE system an unattractive candidate in practice. In this study, we aim to explore the limiting factors stopping PURE cell-free reactions and propose methods to increase the efficacy of the system.

Shota Nishikawa

Shota Nishikawa*, Po-Hsiang Wang, Shawn McGlynn, and Kosuke Fujishima

poster number 39

De novo synthesis of iron-sulfur proteins using recombinant SUF system in a cell-free system

Iron-sulfur clusters are essential cofactors mediating single-electron transfer in respiratory and metabolic networks. However, heterologous iron-sulfur protein expression is challenging due to (i) the requirements for iron-sulfur cluster assembly, (ii) the O2 lability of iron-sulfur clusters. Here, we established a facile and efficient protocol to express mature [4Fe-4S] proteins in the reconstituted cell-free translation system (i.e., the PURE system) under aerobic conditions. The incorporation of recombinant SUF helper proteins to the PURE system enabled streamlined mRNA translation and [4Fe-4S] cluster assembly under anaerobic conditions. To reconcile the O2 lability of [4Fe-4S] clusters, we further added an O2-scavenging three-enzyme cascade to the PURE system. In this cascade, formate dehydrogenase, flavin reductase and catalase are used to reduce O2 to water. This cascade also supplies FADH2 to the SUF helper proteins as well as removing O2 from the reactions, allowing a one-pot synthesis of mature [4Fe-4S] proteins under aerobic conditions with a high maturation rate (>90%). Our new approach will expand the repertoire of cell-free biocatalysis, paving the way for future reconstruction of redox-active synthetic cells.

Surendra

Surendra Yadav

poster number 41

Building Synthetic Metabolism to Efficiently Drive the PURE Cell-free system

Cell-free protein synthesis (CFPS) platforms have recently emerged as powerful tools to produce recombinant proteins rapidly and inexpensively, by harnessing biological gene expression inside in vitro biochemical reactions. The Protein Synthesis Using Recombinant Elements (PURE) system combines purified molecular components required for transcription and translation with energetic substrates and cofactors to carry out CFPS. This completely defined system can be rapidly modulated, offers straightforward product purification, and is well-suited to the construction of minimal cells. However, the system is notably limited by low reaction yields and lifetimes (~2h) compared to conventional lysate-based systems. One way to improve reaction performance is to reengineer the biochemical energy regeneration system. The PURE system uses a basic ATP regeneration system based on creatine phosphate, which leads to accumulation of inorganic phosphate, a by-product inhibitory to CFPS at high concentrations. The aim of this project is to explore different synthetic systems to regenerate ATP, starting with a three-enzyme system based on pyruvate oxidase, catalase, and acetate kinase, which was successfully used to control phosphate accumulation in lysates. Our preliminary results show that this system is thermodynamically as well as kinetically feasible in the PURE reaction conditions. The three enzymes have been purified and are being tested in the PURE system for their energy regeneration role. The successful implementation of these new pathways in the PURE system will help improve performance in terms of reaction lifetime and yield.

Kirill Alexandrov

Shayli Varasteh Moradi, Sergey Mureev, Wayne A Johnston, Yue Wu, Patricia Walden, Zhenling Cui and Kirill Alexandrov

poster number 45

Development and applications of a eukaryotic cell-free expression system based on Leishmania tarentolae

Proteins are workhorses of the biotech industry and Life sciences. Yet our ability to create protein-based systems with desired features is woefully underdeveloped. In an effort to create effective tools for rapid protein production and engineering we have exploited the unique biology of Kinetoplastida to develop Leishmania tarentolae-based in vitro expression systems . We present the motivations, opportunities and hurdles that we faced creating and disseminating different versions of the system. The system has been tested in a range of cell-biological, biotechnological and protein engineering projects. We will discuss the lessons learned and show case several examples including the use of Leishmania cell free system to map protein:protein interaction network of SARS-CoV-2 virus. We will offer my perspective on the place of the developed technology in the Life Sciences toolbox.

Vadim Bogatyr

Vadim Bogatyr¹*, Andreas Biebricher¹, Maike Hansen², Roel Maas², Wilhelm Huck², Gijs Wuite¹ 1 – Vrije Universiteit Amsterdam, Amsterdam, the Netherlands 2 – Radboud University, Nijmegen, the Netherlands

poster number 47

Counting mRNAs: towards single-molecule in vitro transcription (smIVT) assay

Transcription is a vital cellular process in which an RNA polymerase produces messenger RNAs (mRNA) from a DNA template. Therefore, on its trajectory toward a viable artificial cell, synthetic biology aims to reproduce transcription reliably and understand how it works in a confined environment. To this end, a number of commercially-available or lab-made transcription systems have been established for in vitro applications. Moreover, using fluorescent probes, researchers could follow live the changes in RNA concentration over time inside giant unilamellar vesicles (GUVs). However, most of these measurements have concerned DNA concentrations far exceeding those in natural cells, where there is usually just a single DNA copy. Here we show our progress towards a single-molecule transcription assay that goes beyond these limitations and allows us to track single DNA and RNA in solution and inside GUVs. We have achieved this with two approaches, both reliant on fluorescent probes that become more fluorescently active after an interaction with the transcript mRNA. In the first one, we use molecular beacons (MB), a 26-base long DNA hairpin modified with ATTO 647N dye and an Iowa Black RQ quencher. Closed and dim in the solution, they open and light up upon binding to the complementary sequence on the mRNA. The template DNA here had 32 MB-complementary repeats to ensure a signal-to-noise ratio sufficient for detecting single mRNAs. In the second approach, we have utilized Spinach aptamer, a specific mRNA sequence that, upon folding, can stabilize a DFHBI dye molecule and enhance its signal. Similarly, to improve detection, 32 repeats of the Spinach sequence were encoded on the DNA construct. We demonstrate these two single-molecule approaches for tracking transcription in real-time at picomolar DNA concentrations, discuss their performance and compare different transcription systems.

Elisabeth Edgerton

Elisabeth Edgerton*, Maddie Westenberg, Wakana Sato, Madilyn Stahl, Ahmed Shahkhan, Akshay Peddi, Dadir Awad, Aaron Engelhart, Katarzyna Adamala

poster number 54

Cellulator: Developing a Validated Computational Model of Cell-free Protein Synthesis

Cell-free protein synthesis is an efficient method for expressing and producing proteins, but predicting yields can be difficult. Computational models can help to predict the impact of varying parameters, but few models exist that have been experimentally validated and include all the necessary biological components. We developed a computational model of cell-free protein synthesis that includes transcription, translation, energy regeneration, and aminoacylation. Experimental kinetic data collected from the literature was used to set parameters for the model, and we ran a series of simulations to predict protein synthesis for the protein HiBiT. To validate the model, we ran experimental trials in which the concentrations of DNA, tRNA, amino acids, and rNTPs were individually varied, and the resulting protein yields were measured. Experimental results demonstrated that the model accurately predicted the changes in protein output when the concentrations of DNA and tRNA were varied. However, when the concentrations of amino acids and rNTPs were varied, the experimental results differed from the model predictions, possibly due to a lack of consideration of magnesium ions in the model. Additionally, the numerical protein output generated by the model was not consistent with the actual protein yields measured in the experiments, which indicates that the model could be improved upon by accounting for additional parameters or further refining the kinetic data. Overall, our computational model provides a valuable tool for predicting the impact of varying system parameters on protein synthesis and could potentially be used to optimize the PURE cell-free system for specific protein production. By comparing the simulation results with experimental data, we can continue to build upon the model and improve its accuracy for further testing.

Christopher Deich

Christopher Deich, Nathaniel Gaut, Brock Cash, Tanner Hoog, Aaron Engelhart, Katarzyna Adamala

poster number 55

Synthetic minimal cell with 90kb genome undergoing natural selection

Assembling a live cell from non-living components is one of the biggest challenges of experimental science. Here we demonstrate a complete cell cycle of a synthetic cell undergoing natural selection: DNA replication, gene expression, genetically encoded growth, and resource acquisition via feeding. To enable growth and feeding, we engineered a genetically encoded mechanism for liposome fusion. The competition between two genomes results in natural selection, where one population feeds and grows faster, producing more offspring. The synthetic cells presented here have fully defined chemical composition, demonstrating minimal chemical recipe for life-like system. Overall, we present a cell cycle coupled with natural selection, that creates a chassis for the evolution of new lineages of life.

Session Minimal cell on live chassis: Mycoplasma and others posters

Daniela Bittencourt

Daniela Matias de C. Bittencourt*, Marco Antônio de Oliveira, Raquel Sampaio de Oliveira, Mariana Mathias C. Araujo, Elibio Rech, John I. Glass

poster number 18

Development of chromosomal DNA-free Mycoplasmas as recipients of functional genetic constructs

The study and development of minimal genomes serve as a basis for better understanding the structural and functional organization of the genome and, consequently, how to manipulate it to perform specific functions in response to external stimuli. Therefore, the development of synthetic cells and effective tools to build them will contribute to more precise genetic regulation and the generation of increasingly innovative processes and products. In this study we report the development of chromosome-free cells based on the minimal cell JCVI-Syn3.0B, as a prototype for the design of synthetic cells capable of acting as biosensors and performing specific functions in response to external stimuli. To this end, we engineered a genetic circuit using serine integrases (INT9) to activate the expression of the endonuclease I-Ceul, which together with endogenous endonucleases, completely degrades the host cell genome. The endonuclease I-Ceul recognizes a specific 26 base pairs sequence (5'-TAACTATAACGGTCCTAAGGTAGCGA-3'), present in most bacterial genomes encoded within the 23S rRNA rrl gene. To analyze the functionality of the new cell (SimCell_JCVI-syn3B), a genetic construct with the complete metabolic pathway of arginine deaminase was also used. Due to the lack of a chromosome, SimCell_JCVI-syn3B is unable to replicate, which lessens the biosafety concerns associated with genetically modified organisms. This approach will not only enable the development of innovative and safe biological inputs for application in the different productive sectors of agriculture, industry and medicine, but it also comes as an alternative method to prepare recipients for synthetic genomes.

Feilun Wu

Feilun Wu, Nacyra Assad-Garcia, Eugenia Romantseva, Elizabeth Strychalski, John Glass

poster number 19

Expanding toolkit for whole genome transplantation

Whole genome transplantation (WGT) is a process of installing an isolated bacterial genome into a suitable recipient cell, resulting in a transplant with the phenotype and genotype of the donor genome. Building cells programmed with synthetic or other radically engineered genomes will be a slow and expensive process until a method like genome transplantation can be used for a wide range of bacteria species. However, currently, whole genome transplantation is only successful in mycoplasma. In order to expand whole genome transplantation beyond mycoplasma, fundamental understanding of the mechanism of whole genome transplantation needs to be acquired, and new techniques and technologies such as automation and microfluidics devices need to be developed. Here we outline our plan for the expansion of whole genome transplantation toolkit.

Ronald Rodriguez

Ronald Rodriguez, John I Glass

poster number 50

Genetic and Physiological Characterization of a Genome Minimized, Synthetic Bacterial Cell

The construction of living cells with minimized genomes required development of methodologies that have been extremely valuable to synthetic biologists performing grand scale genome engineering. JCVI-Syn 3.0 (Syn 3.0) is a synthetic, minimal bacterial cell derived from Mycoplasma mycoides containing only 473 genes and is currently being utilized as a model system to study the basic principles of life. Syn 3.0 can replicate its genome and undergo cell division, despite the absence of genes thought to be necessary for normal cell division. Syn 3.0 cell division generates irregularly shaped cells and filamentous structures through an unknown mechanism. Normal cell division can be restored by the addition of seven non-essential genes, including cell division genes ftsZ, sepF, and five genes with unknown functions. To develop a better understanding of cell division in Syn 3.0, we will examine the cellular localization of these non-essential gene products with high-resolution light microscopy. Bacterial histone-like protein (HupA) is a high-copy essential protein in natural bacteria. HupA copy number per cell decreased from approximately 6000 to 28 in our minimal cell, presumably due to deletion of a non-essential gene located directly upstream of hupA. We hypothesize this is why chromosome conformation capture studies of our minimal cell displays a lack of long-range chromosome interactions. Perhaps by dramatically reducing the copy number of HupA per cell, we have recapitulated a physiological state of primordial cells before the evolution of chromatin. We will restore the non-essential gene upstream of hupA and determine if our long-range chromosomal interactions date is similar to what occurs in other bacteria. Our planned minimal cell experiments should expand our understanding of minimal cell physiology and potentially provide insight into what primordial cells may have looked like before the emergence of modern cell division mechanisms and chromatin in bacteria.

Session Cytoskeleton: protein, DNA, other structures posters

LAURA CASAS FERRER

Casas Ferrer Laura, Xiangting Lei, Honts Jerry, Bhamla Saad

poster number 26

Mechanical behavior of optically controlled motor-free cytoskeletal networks under biomimetic confinement

In the present work we study the behavior of a motor-free cytoskeletal network under physical confinement. Our system is made of the tetrahymena calcium-binding protein 2 (tcb2), which has the ability to assemble into network-like structures and to contract upon increase of free calcium in solution. The network is enclosed inside a cell-sized liposome, which mimics the structure and composition of a cellular membrane. The contraction of these networks is possible thanks to a photolabile compound that chelates calcium and releases it upon irradiation with UV light, which is also enclosed inside the lipid bilayer. We study the response of the protein network upon controlled calcium release within the membrane boundaries, the membrane deformations, and the motion that arises from it. The study of motor-free cytoskeletons is important in the context of bioengineering, since it opens new venues for the design of more energetically efficient systems and devices. The ability to optically control calcium signaling pathways is also a rising field in biomedicine, which has already offered solutions for diagnosis and treatment of various diseases, including cancer, and several muscular and neurological disorders. The convergence of these two disciplines in the presented work will contribute to building a solid ground for the development of the next generation of optically-controlled bioactuators and biosensing devices.

María Reverte López

María Reverte-López*, Marion Jasnin, Petra Schwille

poster number 28

Positioning of actomyosin rings and vesicle blebbing mediated by the MinDE system

One of the challenges of bottom-up synthetic biology is the engineering of a minimal set of modules for self-division of synthetic cells. To produce the contractile force required for the excision of cell-like compartments such as lipid vesicles, reconstituted cytokinetic rings made of actin are considered the most promising constituent of a potential synthetic division machinery. However, control over the assembly and localization of actomyosin rings inside giant unilamellar vesicles (GUVs) is still required in order to reach effective mid-vesicle constriction. Here, we show the combined in vitro reconstitution within GUVs of actomyosin rings and a synthetic positioning module: the bacterial MinDE protein system, a versatile tool that allows the active transport of any diffusible cargo molecule on membranes. Incorporating Min oscillations to actin-containing GUVs induces the stacking and localization of actin rings at the equatorial plane of lipid vesicles. Remarkably, the synergistic effect of Min oscillations with the contractile nature of actomyosin bundles induces mid-vesicle membrane deformations and blebbing, which lead to the breaking of spherical GUV symmetry and dramatic outward membrane expansion. Our system thus demonstrates that it is possible to integrate synthetic modules dissimilar in nature, and showcases how emerging effects arising from their joint reconstitution could contribute to the development of a synthetic division toolset.

Tuesday, May 23 sessions and keynotes

Tuesday, May 23, breakout sessions

Containers #3

Tuesday, May 23, Johnson room, 1:00PM - 2:30PM
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Applications #3

Tuesday, May 23, Ski-U-Mah room, 1:00PM - 2:30PM
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Building Blocks #3

Tuesday, May 23, Swain room, 1:00PM - 2:30PM
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Containers #4

Tuesday, May 23, Johnson room, 3:00PM - 4:30PM
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Applications #4

Tuesday, May 23, Ski-U-Mah room, 3:00PM - 4:30PM
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Building Blocks #4

Tuesday, May 23, Swain room, 3:00PM - 4:30PM
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Containers #5

Tuesday, May 23, Johnson room, 4:45PM - 6:00PM
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Cell-free #1

Tuesday, May 23, Ski-U-Mah room, 4:45PM - 6:00PM
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Building Blocks #5

Tuesday, May 23, Swain room, 4:45PM - 6:00PM
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Tuesday, May 23, keynote lectures
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Dora Tang

Tuesday, May 23, Memorial Hall, 9:15AM - 10:00AM

Using bottom up approaches to unravel the chemical-physico principles of life

One of the goals of bottom-up synthetic biology is to build living cells from scratch. Biology is well equipped in exploiting a large number of out-of-equilibrium processes to support life. A complete understanding of these mechanisms is still in its infancy due to the complexity and number of the individual components involved in the reactions. However, a bottom-up approach allows us to replicate key biological processes using a small number of basic building blocks. Moreover, this methodology has the added advantage that properties and characteristics of the artificial cell can be readily tuned and adapted.
In this talk, I will provide an overview of the strategies we adopt in our lab to build living systems from scratch that rely on compartmentalisation as the defining feature to support out-of-equilibrium behaviour. Specifically, I will talk about the design and synthesis of artificial cells based on liquid-liquid phase separation (coacervation) and hydrophobic effects such as lipid vesicles and proteinosomes and describe how these compartments may be used as platforms for implementing dynamic biological behaviours including: RNA catalysis and communication. I propose that our bottom-up approaches are effective in establishing living systems from scratch and in doing so provide unique model systems that can help to unravel the physico-chemical principles of living systems.

Sebastian Maerkl

Tuesday, May 23, Memorial Hall, 10:00AM - 10:45AM

On Biochemical Constructors and Synthetic Cells

Cell-free synthetic biology emerged as a viable in vitro alternative for biological network engineering. Cell-free synthetic biology implements biological systems in a coupled transcription – translation reaction and therefore is a well-defined environment that is easier to control and interrogate than complex cellular systems. I will discuss several technological and methodological advances including the development of microfluidic chemostat devices, a high-throughput microfluidic device, and a method to easily produce a recombinant cell-free system. With these tools we were able to rapidly prototype genetic networks and transplant them into living hosts and engineered gene regulatory networks from the bottom-up with synthetic Zinc-finger transcriptional regulators. More recently, we were able to demonstrate that it is possible to create a partially self-regenerating cell-free system that continuously produces up to seven essential proteins and does so for an extended period of time. This work lays the foundation for the development of a universal biochemical constructor and may ultimately enable the creation of a synthetic cell.

Tuesday, May 23 conference dinner

Wednesday, May 24 sessions and keynotes

Wednesday, May 24, breakout sessions

Cell-Free Systems #2

Wednesday, May 24, Ski-U-Mah room, 1:00PM - 3:00PM
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Building Blocks #6

Wednesday, May 24, Swain room, 1:00PM - 3:00PM
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Wednesday, May 24, keynote lectures
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Paul Freemont

Wednesday, May 24, Memorial Hall, 9:15AM - 10:00AM

Constructing synthetic cells – Biology’s 21st century grand challenge

The construction of synthetic cells constitutes one of the greatest scientific challenges of the 21st century and involves researchers from many disciplines including chemistry, physics, biology, engineering and maths to name a few. This intensely interdisciplinary activity confronts the fundamental nature of living systems by attempting to construct a synthetic cell with the properties of life. Through such a grand challenge we will not only learn about the detailed molecular functions of living systems but also extend the materiality of living systems to elements not found in life. I will discuss different approaches to address this grand challenge and also introduce the role of biofoundries to enable systematic measurement and reproducible construction providing access for many more researchers to join the field. I will also cover our recent work on cell-free engineering of extracellular vesicles and introduce the concept of hybrid synthetic cells.

John Glass

Wednesday, May 24, Memorial Hall, 10:00AM - 10:45AM

Design, Construction, and Analysis of a Synthetic Minimal Bacterial Cell

The minimal cell is the hydrogen atom of cellular biology. Such a cell, because of its simplicity and absence of redundancy, would be a platform for investigating just what biological components are required for life, and how those parts work together to make a living cell. Since the late 1990s, our team at the Venter Institute has been developing a suite of synthetic biology tools that enabled us to build what previously has only been imagined, a minimal cell. Specifically, a bacterial cell with a genome that expresses only the minimum set of genes needed for the cell to divide every two hours that can be grown in pure culture. That minimal cell has about half of the genes that are in the bacterium on which it was based, Mycoplasma mycoides JCVI syn1.0, the so-called synthetic bacteria we reported on in 2010. We used transposon bombardment to identify non-essential genes, and genes needed to maintain rapid growth in M. mycoides. Those findings required re-design and re-synthesis of some reduced genome segments. Three cycles of design, synthesis, and testing, with retention of quasi‐essential genes, produced synthetic bacterium JCVI‐Syn3.0 (531 kb, 474 genes), which has a genome smaller than that of any autonomously replicating cell found in nature. Synthetic bacterium JCVI-Syn3.0 retains almost all genes involved in synthesis and processing of macromolecules. Surprisingly, it also contained 149 genes with unknown biological functions, suggesting the presence of undiscovered functions essential for life. This minimal cell is a versatile platform for investigating the core functions of life, and for exploring whole‐genome design. Since it was initially reported in 2016, we have identified functions for about 50 of the original 149 genes of unknown function. These findings have been used to create flux balance analysis and kinetic whole cell computational models of our minimal cell that replicate laboratory observations about our minimal cell. Additionally, we have used JCVI-syn3.0, which has an abnormal cell division and cell morphology phenotype, and a JCVI-syn3.0 mutant containing an additional seven non-essential genes that has divides normally and looks like wild type Mycoplasma mycoides to investigate how modern cell division and cell size control might have evolved.

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