Orthogonal Light-Dependent Membrane Adhesion Induces Social Self-Sorting and Member-Specific DNA Communication in Synthetic Cell Communities

Developing orthogonal chemical communication pathways in diverse synthetic cell communities is a considerable challenge due to the increased crosstalk and interference associated with large numbers of different types of sender-receiver pairs. Herein, the authors control which sender-receiver pairs communicate in a three-membered community of synthetic cells through red and blue light illumination. Semipermeable protein-polymer-based synthetic cells (proteinosomes) with complementary membrane-attached protein adhesion communicate through single-stranded DNA oligomers and synergistically process biochemical information within a community consisting of one sender and two different receiver populations. Different pairs of red and blue light-responsive protein-protein interactions act as membrane adhesion mediators between the sender and receivers such that they self-assemble and socially self-sort into different multicellular structures under red and blue light. Consequently, distinct sender-receiver pairs come into the signaling range depending on the light illumination and are able to communicate specifically without activation of the other receiver population. Overall, this work shows how photoswitchable membrane adhesion gives rise to different self-sorting protocell patterns that mediate member-specific DNA-based communication in ternary populations of synthetic cells and provides a step towards the design of orthogonal chemical communication networks in diverse communities of synthetic cells

Minimally complex nucleic acid feedback control systems for first experimental implementations

Chemical reaction networks based on catalysis, degradation, and annihilation may be used as building blocks to construct a variety of dynamical and feedback control systems in Synthetic Biology. DNA strand-displacement, which is based on DNA hybridisation programmed using Watson-Crick base pairing, is an effective primitive to implement such reactions experimentally. However, experimental construction, validation and scale-up of nucleic acid control systems is still significantly lagging theoretical developments, due to several technical challenges, such as leakage, crosstalk, and toehold sequence design. To help the progress towards experimental implementation, we provide here designs representing two fundamental classes of reference tracking control circuits (integral and state-feedback control), for which the complexity of the chemical reactions required for implementation has been minimised. The supplied ‘minimally complex’ control circuits should be ideal candidates for first experimental validations of nucleic acid controllers

DNA-based communication in populations of synthetic protocells

Developing molecular communication platforms based on orthogonal communication channels is a crucial step towards engineering artificial multicellular systems. Here, we present a general and scalable platform entitled ‘biomolecular implementation of protocellular communication’ (BIO-PC) to engineer distributed multichannel molecular communication between populations of non-lipid semipermeable microcapsules. Our method leverages the modularity and scalability of enzyme-free DNA strand-displacement circuits to develop protocellular consortia that can sense, process and respond to DNA-based messages. We engineer a rich variety of biochemical communication devices capable of cascaded amplification, bidirectional communication and distributed computational operations. Encapsulating DNA strand-displacement circuits further allows their use in concentrated serum where non-compartmentalized DNA circuits cannot operate. BIO-PC enables reliable execution of distributed DNA-based molecular programs in biologically relevant environments and opens new directions in DNA computing and minimal cell technology

Noise Minimization in Cell-Free Gene Expression

Biochemical reactions that involve small numbers of molecules are accompanied by a degree of inherent randomness that results in noisy reaction outcomes. In synthetic biology, the ability to minimize noise particularly during the reconstitution of future synthetic protocells is an outstanding challenge to secure robust and reproducible behavior. Here we show that by encapsulation of a bacterial cell-free gene expression system in water-in-oil droplets, in vitro-synthesized MazF reduces cell-free gene expression noise &gt;2-fold. With stochastic simulations we identify that this noise minimization acts through both increased degradation and the autoregulatory feedback of MazF. Specifically, we find that the expression of MazF enhances the degradation rate of mRNA up to 18-fold in a sequence-dependent manner. This sequence specificity of MazF would allow targeted noise control, making it ideal to integrate into synthetic gene networks. Therefore, including MazF production in synthetic biology can significantly minimize gene expression noise, impacting future design principles of more complex cell-free gene circuits.</p

Engineering cytokine therapeutics

Cytokines have pivotal roles in immunity, making them attractive as therapeutics for a variety of immune-related disorders. However, the widespread clinical use of cytokines has been limited by their short blood half-lives and severe side effects caused by low specificity and unfavourable biodistribution. Innovations in bioengineering have aided in advancing our knowledge of cytokine biology and yielded new technologies for cytokine engineering. In this Review, we discuss how the development of bioanalytical methods, such as sequencing and high-resolution imaging combined with genetic techniques, have facilitated a better understanding of cytokine biology. We then present an overview of therapeutics arising from cytokine re-engineering, targeting and delivery, mRNA therapeutics and cell therapy. We also highlight the application of these strategies to adjust the immunological imbalance in different immune-mediated disorders, including cancer, infection and autoimmune diseases. Finally, we look ahead to the hurdles that must be overcome before cytokine therapeutics can live up to their full potential

DNA storage in thermoresponsive microcapsules for repeated random multiplexed data access

In support of the publication "DNA storage in thermoresponsive microcapsules for repeated random multiplexed data access" we share the following datasets and code: AutoCAD drawing of the microfluidic trapping device. Sequences of the DNA used to encode the 25 files used in the current study. FASTQ-files of the sequencing experiments of Figures 5b and d. Python scripts that allow for the reproduction of our sequencing data analysis. The code has been tested on MacOS 13.0.1, Python 3.7.13, samtools 1.16.1 and BWA 0.7.17

Dynamic modulation of proximity-induced enzyme activity using supramolecular polymers

Contains fulltext : 221463.pdf (publisher's version ) (Open Access

Protocellular CRISPR/Cas-Based Diffusive Communication Using Transcriptional RNA Signaling

Protocells containing enzyme-driven biomolecular circuits that can process and exchange information offer a promising approach for mimicking cellular features and developing molecular information platforms. Here, we employ synthetic transcriptional circuits together with CRISPR/Cas-based DNA processing inside semipermeable protein-polymer microcompartments. We first establish a transcriptional protocell that can be activated by external DNA strands and produce functional RNA aptamers. Subsequently, we engineer a transcriptional module to generate RNA strands functioning as diffusive signals that can be sensed by neighboring protocells and trigger the activation of internalized DNA probes or localization of Cas nucleases. Our results highlight the opportunities to combine CRISPR/Cas machinery and DNA nanotechnology for protocellular communication and provide a step towards the development of protocells capable of distributed molecular information processing

A Theoretical Framework for Implementable Nucleic Acids Feedback Systems

Chemical reaction networks can be utilised as basic components for nucleic acid feedback control systems’ design for Synthetic Biology application. DNA hybridisation and programmed strand-displacement reactions are effective primitives for implementation. However, the experimental validation and scale-up of nucleic acid control systems are still considerably falling behind their theoretical designs. To aid with the progress heading into experimental implementations, we provide here chemical reaction networks that represent two fundamental classes of linear controllers: integral and static negative state feedback. We reduced the complexity of the networks by finding designs with fewer reactions and chemical species, to take account of the limits of current experimental capabilities and mitigate issues pertaining to crosstalk and leakage, along with toehold sequence design. The supplied control circuits are quintessential candidates for the first experimental validations of nucleic acid controllers, since they have a number of parameters, species, and reactions small enough for viable experimentation with current technical capabilities, but still represent challenging feedback control systems. They are also well suited to further theoretical analysis to verify results on the stability, performance, and robustness of this important new class of control systems. Keywords: feedback control; chemical reaction networks; nucleic acids; strand displacement circuits; synthetic biolog