Invited speakers
Nediljko Budisa,
Top-down approach to synthetic life through directed evolution of synthetic bacterial cells with alternative genetic codes.
Life on Earth is a single entity thanks to the universal genetic code, i.e., the genetic code is basically the same for all organisms – all living beings use the same “genetic language”. This biological framework enables the translation of the genetic message written in DNA into life-sustaining proteins consisting of 20 canonical amino acids. Why “only” 20? Clearly, the reasons for the selection of these 20 amino acids in the genetic code remain a mystery.
Recently, we proposed the Alanine World Model, which provides insight into how amino acid monomers are selected for protein building within the genetic code’s repertoire, using a peptide and protein chemistry perspective. By incorporating the RNA -World Hypothesis (along with the Hartman-Smith model) and Co-evolution theory, our Alanine World Model presents a credible explanation for the chemical origin of the genetic code’s amino acid repertoire.
The most effective way to confirm the validity of these models is to create synthetic cells, or parallel biological worlds, that exhibit variations in their morphology, chemistry, metabolism, energy, and information transfer from traditional life forms. Furthermore, these cells ought to possess genetic codes that are entirely distinct from those found in life as we know it.
In my talk, I will not only give an overview of the Big picture, but also provide brief insights into our recent top-down exploration of adaptive lab evolution and code engineering, which could serve as steps towards achieving these goals.
Barbara Di-Ventura
University of Freiburg, Germany
Inteins as molecular tools for synthetic biology
Inteins are unique proteins that are capable of self-catalyzed excision from precursor proteins and the subsequent joining of the flanking sequences (exteins) with a native peptide bond. This remarkable post-translational modification occurs through a series of nucleophilic attacks and peptide rearrangements, resulting in the removal of the intein and the ligation of the exteins.
Inteins can be found in nature as either contiguous or split proteins. Contiguous inteins are characterized by uninterrupted sequences, where the intein itself is flanked by the N- and C-terminal exteins. Split inteins are divided into two or more segments that are encoded by separate DNA sequences; therefore, the N- and C-exteins are initially physically separated. Split inteins require the association of multiple subunits (most often two, but we and others have shown that inteins can be split in three fragments and still maintain activity) to function. Split inteins can be strategically engineered to facilitate protein trans-splicing, enabling the controlled assembly of proteins made of predefined domains or the site-specific modification of target proteins. The distinct characteristics of contiguous and split inteins provide researchers with a diverse range of tools to manipulate and engineer proteins for various applications in synthetic biology.
In this talk, I will first provide an overview of inteins and their fundamental properties, and how they can be harnessed as powerful tools in the laboratory.
I will then highlight two examples of how our research group has utilized inteins as molecular tools, namely for protein circularization and for selection of cell populations positive for two distinct plasmids using a single antibiotic.
In the final part of the talk, I will introduce a web server called Int&in we recently developed, which allow users to run a machine learning algorithm to predict novel functional split sites within any intein of choice. I will show how this algorithm facilitated the engineering of novel light-inducible versions of gp41-1, the most efficient intein to date.
Ramez Daniel
Technion – Israel Institute of Technology, Israel
Synthetic Biology – where computer logic meets cell biology
The design of biological computers interacting with bio-compounds for cellular control, processing, storage, and actuation using gene circuits inside living cells is gaining prominence due to their capabilities for solving significant challenges in biotechnological and medical applications. For example, synthetic gene circuits for cancer therapy are required to perform sophisticated tasks such as targeting malignant cells, avoiding damage to healthy cells and overmedication, and adapting to cell heterogeneity and environmental changes. Such systems often include sensory systems that receive gradual signals combined with decision-making capability for drug release. In this seminar, I will review the current state of gene circuits, with some of its applications and its technical challenges. Then, I will show our recent study, which describes the design principles of genetically encoded analog-to-digital converters (ADC) in living cells. The ADC circuit can be easily reconfigured to other functions and is useful for various intracellular and extracellular biosensing applications as a ternary switch with distinct low, medium, and high output states. Finally, I will describe a new computing approach that allows the implementation of artificial neural networks in living cells.
Edward Lemke
Gutenberg University, Meinz, Germany
Tools to Decode Molecular Plasticity in the Dark Proteome
Intrinsically disordered proteins (IDPs) account for up to 30% of the eukaryotic proteome. Their polymer-like nature makes them very hard to study by conventional approaches. I will show in my talk how we combine modern synthetic and chemical biology tools with advanced biophysical measurements to develop a path toward studying the protein conformations of IDPs in situ. In my talk, I will focus on the function of IDPs that are central to the nuclear pore complex (NPC) function. Nuclear transport receptors (NTRs) can move through the NPC’s central channel, which is filled with hundreds of phenylalanine-glycine-rich and disordered nucleoporins (FG-Nups), reaching millimolar concentrations with elusive conformational plasticity. Since site-specific labeling of proteins with small but highly photostable fluorescent dyes inside cells remains the major bottleneck for directly studying protein dynamics in the cellular interior, we have now developed a semi-genetic strategy based on novel artificial amino acids that are easily and site-specifically introduced into any protein by the natural machinery of the living cell via a newly developed thin-film synthetic organelle. Our strategy basically equips the living cell with up to three genetic codes and shows the power of biological engineering for rewriting a process as complex as translation inside the living cell, without altering canonical host translation. This allowed us to develop an experimental approach combining site-specific fluorescent labeling of IDPs in non-fixed cells with fluorescent lifetime imaging microscopy (FLIM) to decipher the plasticity of FG-Nups via FRET directly. Our study enabled a conformational look at the densely packed IDPs in the sub-resolution cavity of the NPC and described the conformations of FG-Nups at their functional status as well as the solvent quality in the inner NPC environment.
Yu M, Heidari M, Mikhaleva S, Tan PS, Mingu S, Ruan H, Reinkermeier CD, Obarska-Kosinska A, Siggel M, Beck M, Hummer G, Lemke EA. Deciphering the conformations and dynamics of FG-nucleoporins in situ, Nature. 2023 May;617(7959):162-169.
Reinkemeier CD, Lemke EA. Dual film-like organelles enable spatial separation of orthogonal eukaryotic translation. Cell. 2021 Sep 16;184(19):4886-4903.e21
Reinkemeier CD, Estrada Girona G, Lemke EA, 2019 Designer membraneless organelles en-able codon reassignment of selected mRNAs in eukaryotes. Science, Mar 29;363(6434)
Lianet Noda-Garcia
The Hebrew University of Jerusalem, Israel
Engineering and Optimizing Plastic Biodegradation in Bacteria
Plastic pollution has become a significant environmental issue globally, leading to the development of biodegradation strategies as a potential solution. Some bacterial cells and enzymes have emerged as promising candidates for plastic degradation, but their efficiency needs improvement. Here, I’ll present the latest advances of my laboratory in engineering bacterial strains for plastic biodegradation through rational and semi-rational approaches, like adaptive laboratory evolution. Specifically, I’ll present our advances in developing, by rational metabolic and protein engineering, Pseudomonas putida strains capable of utilizing the polymers polyethylene terephthalate (PET) and polyethylene (PE). Also, I’ll describe our advances in optimizing Rhodococcus ruber C208, a wild-type bacteria capable of degrading oxy-functionalized PE using adaptive laboratory evolution.
Karen Polizzi
Imperial College, UK
Integrating Living Analytics into Biological Manufacturing Processes
Cells have evolved high sensitive and specific mechanisms for sensing the world around them and adapting to changes in the environment. These mechanisms can be co-opted into useful devices for biomanufacturing that have advantages over traditional analytical technologies. However, using these ‘living analytics’ involves several practical considerations around their deployment. How can we prevent them from disturbing the manufacturing process? How can we ensure they function robustly and reproducibly? In this talk, I will discuss our ongoing work in this area, specifically the encapsulation of living biosensors in materials to create a living device that can be used in the monitoring of recombinant protein production processes.
Ariel Lindner
Université de Paris
In vivo synthetic RNA-based liquid-liquid phase separation
Tinkering with bacterial biochemistry is limited by it homogeneous organelle-lacking intracellular environment. We exploited RNA-driven liquid-liquid phase separation (LLPS), borrowed from human disease (Huntingtin), to engineer modular Transcriptionally Engineered Addressable RNA Solvent droplets (TEARS). Such TEARS may be exploited for isolation of biochemical pathways, controlling metabolic branch points, buffering mRNA translation rates and scaffolding protein-protein interactions. We anticipate TEARS to be a simple and versatile tool for spatially controlling E. coli biochemistry.
Stephen Wallace
University of Edinburgh, Scotland, UK
Engineered Microorganisms for Sustainable Chemical Synthesis
Polyethylene terephthalate (PET) is a polyester thermoplastic used extensively across the food and drink, pharmaceutical and cosmetics packaging and textiles industries. Of the >30 M ton PET produced annually, >80% is designed to be single use, resulting in ~24 M ton post-consumer PET waste every year. Although new biocatalytic methods to recycle PET are being investigated, sustainable and scalable methods to upcycle PET waste into value-added chemicals using biotechnology are less developed. In my talk I will outline how our lab is using modern engineering biology methods to design bacteria capable of valorizing post-consumer and industrial PET waste into new products such as materials, pharmaceuticals and flavouring compounds. This will include the use of PET to make vanillin[1] but will focus predominantly on other valuable targets accessed via new multi-step enzymatic pathways from the PET monomer terephthalic acid in E. coli. Together, this work substantiates the philosophy that plastic may be viewed not as a ‘waste’ product but rather as a carbon feedstock for industrial biotechnology from which valuable products can be derived.
[1] Green. Chem. 2021, 23, 4665-4672
Wilfried Weber
INM – Leibniz Institute for New Materials, Saarbrücken, Germany
Molecular Optogenetics – Programming Cells and Materials with Light
Molecular optogenetic technologies allow the control of cellular signaling processes along the whole signal transduction cascade with unmatched spatial and temporal resolution.
Based on an overview of molecular photoreceptors, we will present three aspects of our work: First, we will present extracellular optogenetic strategies to dynamically modulate biological and mechanical properties of the extracellular matrix. Here, we demonstrate that the functional coupling of photoreceptors to chemical polymers, biomolecules and surfaces allows the control of key features of matrix-cell interactions.
We further develop the concept of engineering intracellular liquid materials comprising synthetic or natural transcription factors to adjust transgene activity. We describe different approaches for the stimulus-inducible formation of liquid transcription factor condensates and demonstrate that these colocalize with target promoters and yield a several-fold increased transgene activity compared to the non-engineered transcription factor. We demonstrate that this concept can be applied to different transcription factors to increase target gene activity in cell culture and in mice.
Finally, we will present recent work on engineering viral transduction systems with optogenetic tools to optically guide gene transfer. We demonstrate that this technology allows spatially and temporally controlled gene transfer in primary cells and cell lines. We further demonstrate optically guided transduction at single-cell resolution
Basile I. M. Wicky
University of Washington, Seattle, USA
De novo interactomes for biomolecular computations
Our ability to programme and interface with cellular pathways would greatly benefit from compact circuits composed of fully synthetic components capable of rapid computations directly at the protein level (Gao et al. 2018, Fink et al. 2019, Chen et al. 2020, Chen et al. 2022). In addition, using computational paradigms that harness promiscuous connectivities should improve the information theoretic aspect of biomolecular computations (Antebi et al. 2017, Su et al. 2022).
In this talk I will describe an approach to biomolecular programming that uses computational protein design to generate de novo interactomes. Within this framework, sets of designed proteins that can interact with each other with varying degree of promiscuity are assembled into networks to establish biomolecular equivalents to Boltzmann machines. I will discuss the design and characterisation of the network components, as well as the implementations of Boolean functions in mammalian cells using de novo designed proteins.