Agenda

Synthetic biology is the design of novel or improved biological systems using engineering principles. It is a rapidly growing area with broad applications in medical, chemical, food, and agricultural industries. In this talk, I will give a brief overview of the major milestones in the synthetic biology field in the past two decades and highlight our recent work in the development and application of novel foundational synthetic biology tools. Specifically, I will introduce four interrelated stories, including: (1) development of the Illinois Biological Foundry for Advanced Biomanufacturing (iBioFAB) for next-generation synthetic biology applications; (2) development of new strategies and tools for discovery of novel natural products from genomes and metagenomes; (3) development of genome-scale engineering tools for rapid metabolic engineering applications; and (4) development of new genome engineering tools for human gene therapy and fundamental studies of cell biology.

Lightning talks to advertise posters.

Lightning talks to advertise posters.

• Prof. Tae Seok Moon, Washington University in St. Louis "Multi-input CRISPR-based kill-switches for engineered microbes"

Probiotics are effective chassis for diagnostic and therapeutic applications. However, there are safety concerns associated with using genetically engineered organisms for medical applications. To address these issues, we engineered the probiotic Escherichia coli Nissle to survive only when and where it is needed using CRISPR-based kill-switches (CRISPRks). We first designed a CRISPRks that induces cell death by expressing Cas9 and genome-targeting guide RNAs (gRNAs) in response to a chemical inducer. This design allows cell killing to occur while the microbe is in the gut in response to oral administration of the chemical. We optimized the efficiency and stability of the CRISPRks, achieved more than a 9-log reduction in cell number, and demonstrated genetic stability for up to 28 days of continuous growth. This high killing efficiency was maintained in vivo, where we achieved complete elimination of the probiotic after oral administration of the inducer. To our knowledge, this is the first time on-demand elimination of an engineered microbe that has been demonstrated in vivo. We next modified our chemically inducible-CRISPRks to also induce cell death in response to ambient temperatures below 33’C. This design induces cell killing either in response to oral administration of the chemical or when the microbe is excreted from the body in response to the reduced environmental temperature. This circuit achieved more than a 9-log or 7-log reduction in cell number after exposure to the chemical inducer or temperature downshift, respectively. Our CRISPRks strategy provides a benchmark for future microbial biocontainment circuits. The sensor and killing mechanism are well characterized and functional in many microbes, allowing the CRISPRks design to be broadly applicable. In addition, the temperature-sensing module can be easily replaced with sensors that recognize alternative signals, enabling generalizable kill-switches for applications beyond engineered probiotics.

• Deon Ploessl, Iowa State University, " A Repackaged CRISPR/Cas9 Platform Recasts Non-Homologous End Joining As a Beneficial Instrument in Nonconventional Yeast Engineering"

Inefficient homology-directed repair (HDR) constrains CRISPR/Cas9 genome editing in organisms that preferentially employ non-homologous end joining (NHEJ) to fix DNA double-strand breaks (DSBs). Current strategies to alleviate NHEJ proficiency involve NHEJ disruption. To confer precision editing without NHEJ disruption, we identified shortcomings of conventional CRISPR platforms and developed a novel CRISPR platform, Lowered Indel Nuclease system Enabling Accurate Repair (LINEAR), which drastically enhanced HDR rates (67-100%) compared to previous reports using conventional platforms in four NHEJ-proficient yeasts. With NHEJ preserved, we demonstrate its ability to survey genomic landscapes, identifying loci whose spatiotemporal genomic architectures yield favorable expression dynamics for heterologous pathways. We present a case study that deploys LINEAR precision editing and NHEJ-mediated random integration to rapidly engineer and optimize a microbial factory to produce (S)-norcoclaurine. Taken together, this work demonstrates how to leverage an antagonizing pair of DNA DSB repair pathways to expand the current collection of microbial factories.

• Mark Kathol, University of Nebraska - Lincoln, "Investigating the role of pBBR1’s mobilization protein in plasmid maintenance in non-model bacteria "

Stable plasmid maintenance is critical for testing and employing programmable biological devices in microorganisms. While maintenance mechanisms are well-known for commonly used vectors in model microorganisms, the same is not always true for replicons when utilized in non-model bacteria. . Here we share our work to elucidate plasmid maintenance mechanisms in the non-model Rhodopseudomonas palustris, an extremely metabolically versatile soil bacterium known for its utilization of all four forms of metabolism, its ability to consume recalcitrant feedstocks such as lignin breakdown products, and its ability to produce hydrogen. In R. palustris, the plasmid pBBR1, which contains a mobilization protein, Mob, has been discovered to be required for maintenance. Mobilization proteins are relaxases that nic plasmid DNA at a “nic” site on the plasmid, facilitating conjugation between bacteria through the type IV secretion system by nicking DNA before conjugation and rejoining DNA afterwards. pBBR1’s Mob and the mobilization protein from pMV158, a promiscuous streptococcal plasmid, share a very similar amino acid sequence. The nicking behavior of pMV158 has been experimentally shown to be severely impaired by replacing the catalytic histidine and other active site residues. Using an analogous approach, the effect of the active site mutations on plasmid relaxation and subsequent maintenance will be presented in R. palustris. Interestingly, Mob does not seem to be consistently needed for pBBR1 in all bacterial species. This has been previously documented by successful employment of the plasmid lacking Mob in the pentose, and hexose-consuming bacterium, Paraburkholderia sacchari LMG 19450 LFM101. The seemingly inconsistent role of Mob in plasmid maintenance across species has sparked a parallel investigation in P. sacchari, which will be presented as well. This study provides important groundwork for harnessing the extraordinary biochemical capabilities of non-model bacteria.

• Jihwan Lee, Rice University/Baylor College of Medicine, "Equalizer Expression System Enables Uniform Protein Expression Levels in Transiently Transfected Cells."

The functional study of natural genes and the expression of synthetic circuit actuators generally relies on the expression of these transgenes from plasmids. However, transient plasmid transfections in mammalian cells produce a wide variation of plasmid copy numbers among cells, and consequently cause high expression heterogeneity. This cell-to-cell variability can result in some cells overexpressing at toxic levels while others showing low-expression levels and thereby complicating experiments. Here, we report plasmid-based synthetic circuits -- Equalizers -- that compensate plasmid copy number variation at the single-cell level and provide uniform gene expression in transiently transfected cells. Equalizers combine negative feedback and incoherent feedforward circuitries to moderate the protein expression levels. We demonstrate that Equalizers produce cell-to-cell variation equivalent to the low cell-to-cell variant seen in chromosomally integrated genes and Equalizers operates over a wide range of plasmid copy numbers. We also show that episome-encoded Equalizers enable a rapid generation of extrachromosomal cell lines with stable and uniform expression for over two months. Overall, Equalizers are simple and versatile devices for homogenous gene expression and should facilitate the engineering and study of proteins, cells, and biological circuits.

Lightning talks to advertise posters.

• Prof. Claudia Schmidt-Dannert, U. Minnesota, "Functional protein-based biomaterials"

Self-assembly and self-organization are key principles of biological systems that offer tremendous opportunities for the bottom-up design of functional biomaterials from simple building blocks such as as proteins, nucleic acids, and lipids. Proteins and peptides provide the greatest versatility for the design and low-cost production of supramolecular materials because of their chemical diversity and ability be manufactured recombinantly by cell factories or, in the future, by cell-free expression systems. Protein nanostructures also play important roles in the spatial organization of enzymes at the subcellular level and direct the formation of inorganic-organic composite materials with properties unmatched by synthetic materials. Inspired by these functions, we are harnessing the self-assembling properties of proteins for the design of protein-based nano-architectures for scaffolding of enzymes for biocatalysis and the fabrication of new types of materials. Functionalization of protein-building blocks with additional protein domain and peptide tag fusions allows for control over enzyme attachment and the incorporation of other emergent functions such as biomineralization to create mechanically robust biocomposite materials. In this presentation, I will introduce strategies and examples for the design of such genetically encoded materials.

• Nolan Kennedy, Northwestern University, "Three proteins play critical roles in the self-assembly and function of bacterial microcompartments"

Bacterial microcompartments (MCPs) are protein-based bacterial organelles that enclose metabolic pathways. MCPs help improve metabolic efficiency, providing an advantage for microbes in resource-limited environments. Metabolic engineers have attempted to repurpose MCPs to improve yields of industrially relevant pathways of interest. However, the mechanisms for MCP assembly and function remain unclear. Here, we describe the role of three primary protein components of the MCP shells from Salmonella, providing insight into the assembly mechanism of a model MCP. First, we demonstrate that two proteins, PduA and PduJ, have a unique propensity towards self-assembly and that this property allows these two proteins to stimulate and drive MCP biogenesis. We also developed a novel, high-throughput method that enables rapid analysis of MCP shell protein variants, which yielded exquisite detail on the role of these proteins in MCP shell assembly. These results provided the first demonstration of the essential yet partially redundant role that PduA and PduJ play in MCP shell formation. Furthermore, we demonstrated that a third MCP shell protein, PduB, is not necessary for the formation of MCP shells, a finding that contradicts prior research in the MCP field. Our work demonstrates that MCP shells lacking PduB are smaller and empty, lacking the encapsulated enzymatic core. This implies that while PduB is not essential for MCP shell assembly, it is important for interaction with the enzymatic core. Overall, these findings support a mechanism where PduA and PduJ drive MCP shell assembly. PduB then bridges the gap between the MCP shell and enzymatic core via interactions with PduA and PduJ. These are critical findings for those seeking to re-engineer MCPs for non-native functions. An understanding of the essential components of MCPs, as well as their function in the MCP assembly process, will be necessary to repurpose or design compartments with novel functionalities.

• Angela Chen, University of Texas at Austin, "Engineering Deinococcus radiodurans for Improved Nanoparticle Biosynthesis Using Small RNAs "

Metal nanoparticles possess unique optical and physical properties that have made them invaluable for applications spanning catalysis to antimicrobials. These applications are heavily dependent upon specific properties such as the nanoparticle composition and morphology; however, traditional synthesis methods commonly require energy-intensive processes and toxic reagents. As such, nanoparticle biosynthesis using bacteria has emerged as a promising environmentally-friendly alternative for the production of diverse metal nanoparticles. The inability to control nanoparticle properties due to a lack of mechanistic knowledge though have greatly stymied efforts to improve this process to compete with traditional methods. In this work, we first developed a cell-free process for the synthesis of bimetallic silver and gold nanoparticles using the extremophilic bacterium, Deinococcus radiodurans, and established how nanoparticle properties could be influenced by tuning stress conditions. Building upon the connection between nanoparticle biosynthesis and bacterial stress response, we then propose a role for small RNAs (sRNAs), which are known to be global regulators of stress response, in metal reduction and subsequently nanoparticle formation. Through construction and screening of a library of sRNA deletion strains, we identify four sRNAs that drastically influence biosynthetic silver nanoparticle (AgNP) properties such as the yield, surface chemistry, and overall composition upon deletion, substantiating their importance in the biosynthetic process. Furthermore, the material changes resulting from modulating the expression of these sRNAs lead to enhanced antimicrobial and catalytic activity compared to AgNPs synthesized using the wild-type D. radiodurans strain. Overall, this work illustrates the potential of sRNAs as a new platform for genetically engineering nanoparticle biosynthesis pathways to produce nanoparticles with unique properties and enhanced functionality.

• Cameron Sargent, Washington University in St. Louis, "Synthetic biology enables the microbial polymerization of titin proteins and production of fibers with exceptional mechanical properties "

Natural protein-based and synthetic polymer materials exhibit mechanical properties that make them useful in a variety of applications. However, many of these protein- and petroleum-based materials are plagued by the production issues of low scalability and poor renewability, respectively. If properly harnessed, biology could provide solutions to these challenges and enable the sustainable mass production of many of these materials. While extensive work has been accomplished in engineering genetic circuits and small molecule metabolism, much less progress has been made in the top-down synthetic biology field of creating proteins and polymers for use as materials. One of the most difficult challenges in the biotechnological production of protein-based materials is that the high molecular weight (MW) proteins required to make materials with advantageous performance cannot be produced efficiently in heterologous hosts, being hindered by issues such as genetic recombination, poor translation efficiency, and metabolic burden. To overcome these challenges, we have developed a microbial polymerization platform that employs split inteins to post-translationally assemble ultrahigh MW proteins. Here we report the application of this platform in synthesizing polymers of unprecedented size from the titin protein found in animal muscles. Using an E. coli host, we have expressed and polymerized megadalton titin, which yields high-performance fibers with strength, toughness, and damping capacity that surpass many microbial, natural, and manmade materials. Structural characterization and computational modeling indicate that these titin fibers have unique inter-chain crystallization that enables these outstanding mechanical properties. This novel material has potential applications in fields such as biomedicine and textiles, while the microbial polymerization platform can be used to synthesize a range of protein-based materials with utility across dimensional scales.

Lightning talks to advertise posters.

Poster Session will be in Silverman. Go here for the full list of poster abstracts.

Representatives from academia, industry, and government discuss the challenges and opportunities for commercializing early stage Syn Bio research. Questions welcome!

Panelists

· Nigel Reuel (Iowa State)

· Ross Thyer (Rice)

· Khalid Alam (Stemloop)

· Jesus Soriano Molla (NSF)

· Dan Hussey (UT Austin)

· Weston Kightlinger (SwiftScale Biologics)

Agriculture plays an essential role in our everyday lives, and is a major driver of environmental pollution and greenhouse gas emissions. Synthetic biologists are reimagining how we produce food by applying advanced genetic engineering to enhance crop and livestock productivity, disease resistance, and tolerance to the earth's changing climate. This panel discussion brings together experts from industry and academia that are working at the forefront of agricultural synthetic biology. We will discuss the crucial need for advances in synthetic biology to feed the growing human population, and highlight key opportunities in crop, livestock, and microbial engineering to contribute to the future.

Panelists

• Mark Cigan (Genus PLC)

• Bill Kim (Pairwise)

• Laurie Leonelli (UIUC)

• Gina Neumann (Benson Hill)

• Sergei Svitashev (Corteva)

• Prof. Andy Ellington, UT Austin, "Directed evolution of biosensors for genetic control of metabolism"

The facile identification of genetically encoded biosensors can enables the engineering of metabolism and scaled production, and the development of novel diagnostics. We have developed a unique combined screening and selection approach that quickly refines the affinities and specificities of generalist transcription factors, and using RamR as a starting point we evolve highly specific (>100-fold preference) and sensitive (EC50 <30 μM) biosensors for the alkaloids tetrahydropapaverine, papaverine, glaucine, rotundine, and noscapine. High resolution structures reveal multiple evolutionary avenues for the fungible effector binding site, and the creation of new pockets for different chemical moieties. These sensors further enabled the evolution of a streamlined pathway for tetrahydropapaverine, collapsing multiple methylation steps into a single evolved enzyme. In parallel, we used CamR as a starting point to develop generalist biosensors for a variety of monoterpenes, potentially enabling multiple pathways and production strains to be rapidly screened.

• Kirsten Jung, Northwestern University, "Programming Cell-Free Biosensors with DNA Strand Displacement Circuits"

Cell-free biosensors are emerging as powerful platforms for monitoring human and environmental health. Here, we expand the capabilities of biosensors by interfacing their outputs with toehold-mediated strand displacement circuits, a dynamic DNA nanotechnology that enables molecular computation through programmable interactions between nucleic acid strands. We develop design rules for interfacing biosensors with strand displacement circuits, show that these circuits allow fine-tuning of reaction kinetics and faster response times, build six different types of 2-input logic gates and demonstrate a circuit that acts like an analog-to-digital converter to create a series of binary outputs that encode the concentration range of the target molecule being detected. We believe this work establishes a pathway to create “smart” diagnostics that use molecular computations to enhance the speed and robustness and utility of biosensors.

• Madhumitha Prakash, Purdue University, "Microfluidic Argonaute Mediated Viral Point of Care Diagnostic Device"

The COVID-19 pandemic has strained global diagnostic capacities and highlighted the limitations of conventional lab-based assays, which can take between 1-14 days to receive conclusive results. Current on-site kits have false-negative rates as high as 33%. To provide accurate, non-invasive, affordable, and rapid Point of Care(POC) testing for COVID-19 and other emerging pandemics, Purdue iGEM designed an Argonaute mediated saliva-based viral diagnostic device. The purpose of this project is to study the use of Argonaute proteins found in Thermus thermophilus bacteria (TtAgo) to develop a saliva-based rapid and accurate microfluidic COVID-19 diagnostic device. The diagnostic device works as such: Saliva is inputted into the chip and viral RNA is extracted from it. The RNA is then amplified, converted into double-stranded DNA (dsDNA), and cleaved by TtAgo producing single-stranded DNA fragments(ssDNA). These ssDNA fragments bind to molecular beacons emitting a quantifiable fluorescent signal for conclusive result determination. The team used programming to optimize the biologics of the device, develop CAD models of the microfluidic chip, model the adsorption kinetics of chitosan, and develop a heating circuit for the chip. Throughout the project, the team consulted experts regarding the device’s design and safety and spearheaded an intercollegiate synthetic biology educational initiative. Coupling the biologics of cArgo with chip barcoding and app integration, the team hopes to revolutionize POC diagnostics while making data more accessible for simultaneous viral detection and contact tracing.

• Andrew Hunt, Northwestern University, "Multivalent designed proteins protect against SARS-CoV-2 variants of concern"

Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. In this work we develop multivalent minibinders as potential prophylactic and therapeutic agents to address this problem. We designed multivalent minibinders containing three copies of a minibinder (self-assembled homotrimers), or three linked distinct minibinders (multi-domain fusions) targeting multiple sites on the SARS-CoV-2 spike (S) glycoprotein, geometrically matched to the S protein C3 symmetry and optimized their composition using a rapid cell-free expression and evaluation workflow. The optimized designs have greatly slowed dissociation rates from the S protein, with complex half-lives of more than one week, and restore binding to spike variants that can escape single minibinder monomers. Cryo-EM of the structures reveal that both homotrimer and fusion minibinder constructs likely engage all three RBDs on a single spike protein. The top trimeric and fusion candidates neutralize the wild-type SARS-CoV-2 virus in addition to the B.1.1.7, B.1.351, B.1.1.28, B.1.526, and B.1.617.1 variants with IC50s in the low-to-mid pM range. The top homotrimer candidate additionally provides both pre- and post- exposure therapeutic benefits in a human ACE2-expressing transgenic mouse model. Our approach highlights the utility of computational protein design coupled to rapid experimental prototyping to design potent and mutationally-resilient multivalent inhibitors for pandemic preparedness.

Living organisms use materials in their environment for many purposes, including as building blocks for protective shells and as redox partners to conserve energy. To achieve these functions, living systems form interfaces with materials and use biomolecules to control and monitor the flow of matter across this interface. Inspired by these naturally-occurring systems, my research group seeks to engineer new organisms with tailored abilities to transfer charge and assemble matter across the microbial-material interface.

In the first part of my talk, I will describe how we have engineered bi-directional electronic communication between living microbes and non-living systems using synthetic biology. Our laboratory has pioneered a synthetic biology approach in which we introduce the Mtr extracellular electron transfer pathway from Shewanella oneidensis into non-native microorganisms. This genetic module provides a molecularly-defined path for electrons into and out of a specific redox pool. I will describe how we have used this modular genetic tool to communicate sensing information and to control metabolic rate and gene expression.

In the second part of my talk, I will describe how we programmed bacterial cells to grow into a macroscopic, engineered living material. Programming macroscopic material formation bottom-up from a model organism would offer expansive, user-defined control over material structure and function. When we encoded a high density of weakly interacting proteins on the surface of Caulobacter crescentus, we discovered the cells grow into a centimeter-sized material. Cells within this material remain viable and re-seed growth of new material. This material can be processed into different 3D shapes, can form inorganic-organic composites, and can be programmed to have additional functions. Taken together, this work provides a platform and design rules for creating multifunctional, hierarchically-assembled living materials.