Biology subjects

Maerkl, S. J.

Publications and source records attributed to Maerkl, S. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Trans-acting Determinants of Gene Expression: Effects of Transcription Factor Affinity, Abundance, and Localization

Transcription factors (TFs) regulate gene expression by binding cis-regulatory DNA elements, yet how trans-regulatory characteristics such as TF affinity, concentration, and localization interact with cis-regulatory elements remains largely unclear. We systematically analyzed TF affinity mutants across abundance, and localization states and found that promoter binding-site strength most readily modulated expression levels, followed by TF localization and concentration, while affinity variations were mainly buffered. We further uncover performance trade-offs between TF abundance, localization, and affinity. Together, these results reveal how trans and cis factors collectively shape gene-regulatory output.

systems biology↗

bioRxiv2025

PURE makes PURE: reconstitution of the PURE cell-free system from self-synthesized non-ribosomal proteins

Building a universal biochemical constructor, an autonomously self-replicating bio-chemical system, is a major challenge in synthetic biology. The PURE cell-free system is an ideal starting point for exploring self-regeneration, and its 36 non-ribosomal proteins constitute the primary macromolecular components that must be regenerated. Here, we demonstrate that the PURE system can be reconstituted from proteins synthesized by PURE itself. We first show that each of the 36 non-ribosomal proteins can be individually synthesized in PURE. We then purify the PURE synthesized proteins as pooled subsets and reconstitute a fully functional PURE system by combining the subsets. Finally, we show that all 36 non-ribosomal PURE proteins can be synthesized simultaneously in a single PURE reaction and, after purification, can reconstitute a functional PURE system. Together, these results establish that the non-ribosomal protein components of the PURE system can be self-regenerated, representing a critical step toward the realization of a universal biochemical constructor.

synthetic biology↗

bioRxiv2025

High-throughput cell-free profiling of SARS-CoV-2 RBD variants enables rapid and quantitative in vitro affinity landscape mapping

SARS-CoV-2 variants continue to threaten public health, necessitating the study of cumulative and epistatic effects of receptor-binding domain (RBD) mutations on antibody evasion. We present a high-throughput platform combining cell-free protein synthesis and microfluidics to quantify the affinity of a large number of RBD triplet mutants covering the evolutionary space between wild-type and Omicron against two therapeutic antibodies and one engineered binder. Using rapid in vitro gene assembly and cell-free synthesis, we expressed 518 RBD variants and obtained 31,740 quantitative affinity measurements to generate three comprehensive binding energy landscapes. This approach enables rapid and large-scale in vitro affinity profiling and machine learning-based predictions, providing a valuable tool for studying emerging variants.

systems biology↗

bioRxiv2025

De novo design of phosphorylation-induced protein switches for synthetic signaling in cells

A hallmark of living systems is their ability to respond and adapt to many types of exogenous and endogenous cues. At the core of this capability lie the evolved mechanisms of cellular signaling which perform the relay of biochemical signals throughout the cell. Much of this core signaling functionality in living systems is afforded by the ability of proteins to change their chemical and structural "status", by populating distinct states in conformational landscapes, undergoing post-translational modifications that alter these populations, and dynamically interacting with other cellular partners. Despite the remarkable advances we have witnessed in computational protein design, it remains an outstanding challenge to rationally design many of these aspects that are at the core of biological function. Here, we set out to design a minimal signaling network triggered by phosphorylation, which relies on de novo components that sample low-populated conformational states to control a designed protein interaction. In these designed components we implemented principles of signal amplification, conformational heterogeneity and molecular recognition which are ubiquitously used by nature to sustain biological function in cells. The designed proteins were biochemically and structurally characterized and ultimately are functional in cell-based systems where they regulate the transcription of reporter proteins in a phosphorylation-dependent manner. Overall, this work lays the foundation for designing synthetic signaling cascades using de novo protein components, which could be important to enhance our understanding of natural systems and for relevant applications in synthetic biology.

bioengineering↗

bioRxiv2025

A T7 RNAP regulatory toolbox for cell-free network engineering and biosensing applications

T7 RNA polymerase is ubiquitously used in the fields of synthetic biology and biotechnology. Yet the ability to precisely and modularly regulate T7 RNAP remains surprisingly limited. Here, we developed a T7 RNAP regulatory toolbox consisting of programmable synthetic repressors, activators, and biosensors in a cell-free system. This toolbox enables the scalable design of T7 RNAP based gene regulatory networks and enables the rapid and sensitive detection of diverse biomolecules, including small-molecule drugs, antibodies, and proteins. By integrating a protein design pipeline, we generated biosensors using fully synthetic binders, demonstrating the potential for rapid development of novel protein-based sensors. We constructed a diagnostic cell-free system combining SARS-CoV-2 Spike protein sensing, gene regulatory based amplification, enzymatic amplification, and glucose based detection demonstrating the potential for point-of-care detection with high sensitivity. This work establishes a flexible and expandable framework for constructing gene circuits responsive to a wide range of biomolecules and demonstrates the potential for engineering point-of-care cell-free diagnostic assays.

synthetic biology↗

bioRxiv2025

Continuous in situ synthesis of a complete set of tRNAs sustains steady-state translation in a recombinant cell-free system.

Construction of a self-regenerating biochemical system is critical for building a synthetic cell. An essential step in building a self-regenerative system is producing a complete set of tRNAs for translation, which remains a significant challenge. We reconstituted a complete set of 21 in vitro transcribed (IVT) tRNAs by improving the transcription yield of four tRNAs and optimized their abundance to improve protein yield. Next, we showed that protein expression in the PURE transcription-translation system can be achieved by in situ transcribing tRNAs from 21 linear tRNA templates or a single plasmid encoding all 21 tRNAs. To enable synthesis of mature tRNAs from a circular template, we employed either a nicked plasmid template or T. maritima tRNase Z to post-transcriptionally process the precursor tRNAs. We ultimately achieved continuous in situ synthesis of a complete set of tRNAs capable of supporting sustained, steady-state protein expression in PURE reactions running on microfluidic chemostats. Our findings advance the development of an autopoietic biochemical system.

synthetic biology↗

bioRxiv2024

Long-term protein synthesis with PURE in a mesoscale dialysis system

Cell-free systems are powerful tools in synthetic biology with versatile and wide-ranging applications. However, a significant bottleneck for these systems, particularly the PURE cell-free system, is their limited reaction lifespan and yield. Dialysis offers a promising approach to prolong reaction lifetimes and increase yields, yet most custom dialysis systems require access to sophisticated equipment like 3D printers or microfabrication tools. In this study, we utilized an easy-to-assemble, medium-scale dialysis system for cell-free reactions using commercially available components. By employing dialysis with periodic exchange of the feeding solution, we achieved a protein yield of 1.16 mg/mL GFP in the PURE system and extended protein synthesis for at least 12.5 consecutive days, demonstrating the systems excellent stability.

synthetic biology↗

bioRxiv2024

Towards self-regeneration: exploring the limits of protein synthesis in the PURE cell-free transcription -translation system

Self-regeneration is a key function of living systems that needs to be recapitulated in vitro to create a living synthetic cell. A major limiting factor for protein self-regeneration in the PURE cell-free transcription-translation system is its high protein concentration, which far exceed the systems protein synthesis rate. Here we were able to drastically reduce the non-ribosomal PURE protein concentration up to 97.3% while increasing protein synthesis efficiency. Although crowding agents were not effective in the original PURE formulation, we found that in highly dilute PURE formulations addition of 6% dextran considerably increased protein synthesis rate and total protein yield. These new PURE formulations will be useful for many cell-free synthetic biology applications and we estimate that PURE can now support the complete self-regeneration of all 36 non-ribosomal proteins, which is a critical step towards the development of a universal biochemical constructor and living synthetic cell.

synthetic biology↗

bioRxiv2024

Targeting protein-ligand neosurfaces using a generalizable deep learning approach

Molecular recognition events between proteins drive biological processes in living systems. However, higher levels of mechanistic regulation have emerged, where protein-protein interactions are conditioned to small molecules. Here, we present a computational strategy for the design of proteins that target neosurfaces, i.e. surfaces arising from protein-ligand complexes. To do so, we leveraged a deep learning approach based on learned molecular surface representations and experimentally validated binders against three drug-bound protein complexes. Remarkably, surface fingerprints trained only on proteins can be applied to neosurfaces emerging from small molecules, serving as a powerful demonstration of generalizability that is uncommon in deep learning approaches. The designed chemically-induced protein interactions hold the potential to expand the sensing repertoire and the assembly of new synthetic pathways in engineered cells.

biochemistry↗

bioRxiv2023

C2CAplus: a one-pot isothermal circle-to-circle DNA amplification system

Rolling circle amplification (RCA) is a widely used DNA amplification method that uses circular template DNA as input and produces multimeric, linear single or double stranded DNA. Circle-to-circle amplification (C2CA) has further expanded this method by implementing product re-circularization using restriction and ligation, leading to a higher amplification yield, and enabling the generation of circular products. However, C2CA is a multistep, non-isothermal method, requiring multiple fluid manipulations and thereby compromises several advantages of RCA. Here, we improved C2CA to implement a one-pot, single step, isothermal reaction at temperatures ranging from 25 to 37{degrees}C. Our C2CAplus method is simple, robust, and produces large quantities of product DNA that can be seen with the naked eye.

synthetic biology↗

bioRxiv2023

A field-capable rapid plant DNA extraction protocol using microneedle patches for botanical survey and monitoring

PremiseA novel protocol for rapid plant DNA extractions using microneedles is proposed, which supports botanic surveys, taxonomy and systematics. This protocol can be conducted in the field with limited laboratory skills and equipment. The protocol is validated by conducting sequencing and comparing results with Qiagen spin-column DNA extractions using BLAST analyses. Methods and ResultsTwo sets of DNA extractions were conducted on 13 species spanning various leaf anatomies and phylogenetic lineages: i) fresh leaves were punched with custom polymeric microneedle patches to recover genomic DNA ii) Qiagen DNA extractions. Three plastid (matK, rbcL, trnH-psbA) and one nuclear ribosomal (ITS) DNA regions were amplified, and Sanger or Nanopore sequenced. The proposed method reduced the extraction to 1 min and yielded the same DNA sequences as Qiagen. ConclusionsOur drastically faster and simpler method is compatible with Nanopore sequencing and suitable for multiple applications including high-throughput DNA-based species identification and monitoring.

ecology↗

bioRxiv2022

Improved cell-free transcription-translation reactions in microfluidic chemostats augmented with hydrogel membranes for continuous small molecule dialysis

Increasing protein production capacity of the PURE cell-free transcription-translation (TX-TL) system will be key to implementing complex synthetic biological circuits, and to establish a fully self-regenerating system as a basis for the development of a synthetic cell. Under steady-state conditions, the protein synthesis capacity of the PURE system is likely at least one order of magnitude too low to express sufficient quantities of all PURE protein components. This is in part due to the fact that protein synthesis cant be sustained during the entire dilution cycle, especially at low dilution rates. We developed a microfluidic chemostat augmented with semi-permeable membranes that combines steady-state reactions and continuous dialysis as a possible solution to enhance protein synthesis at steady-state. In batch operation, the continuous dialysis of low molecular weight components via the membranes extended protein synthesis by over an order of magnitude from 2 hours to over 30 hours, leading to a seven-fold increase in protein yield. In chemostat operation, continuous dialysis enabled sustained protein synthesis during the entire dilution cycle even for low dilution rates, leading to six-fold higher protein levels at steady state. The possibility to combine and independently manipulate continuous dialysis and chemostat operation renders our dialysis chemostat a promising technological basis for complex cell-free synthetic biology applications that require enhanced protein synthesis capacity.

synthetic biology↗

bioRxiv2022

Direct encapsulation of biomolecules in semi-permeable microcapsules produced with double-emulsions

Compartmentalization can serve different purposes such as the protection of biological active substances from the environment, or the creation of a unique combination of biomolecules for diagnostic, therapeutic, or other bioengineering applications. We present a method for direct encapsulation of molecules in biocompatible and semi-permeable microcapsules made from low-molecular weight poly(ethylene glycol) diacrylate (PEG-DA 258). Microcapsules are produced using a non-planar PDMS microfluidic chip allowing for one-step production of water-in-PEG-DA 258-in-water double-emulsions, which are polymerized with UV light into a poly-PEG-DA 258 shell. Semi-permeable PEG shells are obtained by adding an inert solvent to the PEG-diacrylate. Due to the favourable hydrophilicity of poly-PEG-DA 258, proteins didnt adsorb to the capsule shell, and we demonstrate the direct encapsulation of enzymes, which can also be dried in the capsules to preserve activity. Finally, we leverage capsule permeability for the implementation of a two-layer communication cascade using compartmentalized DNA strand displacement reactions. This work presents the direct encapsulation of active biomolecules in semi-permeable microcapsules, and we expect our platform to facilitate the development of artificial cells and generating encapsulated diagnostics or therapeutics.

synthetic biology↗

bioRxiv2022

Perfect adaptation achieved by transport limitations governs the inorganic phosphate response in S. cerevisiae.

Cells cope with and adapt to ever-changing environmental conditions. Sophisticated regulatory networks allow cells to adjust to these fluctuating environments. One such archetypal system is the S. cerevisiae Pho regulon. When external inorganic phosphate (Pi) concentration is low, the Pho regulon activates, expressing genes that scavenge external and internal Pi. However, the precise mechanism controlling this regulon remains elusive. We conducted a systems analysis of the Pho regulon on the single cell level under well-controlled environmental conditions. This analysis identified a robust, perfectly adapted Pho regulon state in intermediate Pi conditions, and we discovered a hitherto unknown intermediate nuclear localization state of the transcriptional master regulator Pho4p. The existence of an intermediate nuclear Pho4p state unifies and resolves outstanding incongruities associated with the Pho regulon, explains the observed programmatic states of the Pho regulon, and improves our general understanding of how nature evolves and controls sophisticated gene regulatory networks. We further propose that robustness and perfect adaptation are not achieved through complex network-centric control, but by simple transport biophysics. The ubiquity of multi-transporter systems suggests that similar mechanisms could govern the function of other regulatory networks as well.

systems biology↗

bioRxiv2021

Systematic analysis of low-affinity transcription factor binding site clusters in vitro and in vivo establishes their functional relevance

Transcription factor binding to a single binding site and its functional consequence in a promoter context are beginning to be relatively well understood. However, binding to clusters of sites has yet to be characterized in depth, and the functional relevance of binding site clusters remains uncertain. We employed a high-throughput biochemical method to characterize transcription factor binding to clusters varying across a range of affinities and configurations. We found that transcription factors can bind concurrently to overlapping sites, challenging the notion of binding exclusivity. Furthermore, compared to an individual high-affinity binding site, small clusters with binding sites an order of magnitude lower in affinity give rise to higher mean occupancies at physiologically-relevant transcription factor concentrations in vitro. To assess whether the observed in vitro occupancies translate to transcriptional activation in vivo, we tested low-affinity binding site clusters by inserting them into a synthetic minimal CYC1 and the native PHO5 S. cerevisiae promoter. In the minCYC1 promoter, clusters of low-affinity binding sites can generate transcriptional output comparable to a promoter containing three consensus binding sites. In the PHO5 promoter, replacing the native Pho4 binding sites with clusters of low-affinity binding sites recovered activation of these promoters as well. This systematic characterization demonstrates that clusters of low-affinity binding sites achieve substantial occupancies, and that this occupancy can drive expression in eukaryotic promoters.

biophysics↗

bioRxiv2021

High-throughput single-cell TCR - pMHC dissociation rate measurements performed by an autonomous microfluidic cellular processing unit

We developed an integrated microfluidic cellular processing unit (mCPU) capable of autonomously isolating single cells, perform, measure, and on-the-fly analyze cell-surface dissociation rates, followed by recovery of selected cells. We performed proof-of-concept, high-throughput single-cell experiments characterizing pMHC-TCR interactions on live CD8+ T cells. The mCPU platform analyzed TCR-pMHC dissociation rates with a throughput of 50 cells per hour and hundreds of cells per run, and we demonstrate that cells can be selected, enriched, and easily recovered from the device.

bioengineering↗

bioRxiv2020

CFPU: a cell-free processing unit for high-throughput, automated in vitro circuit characterization

Forward engineering synthetic circuits is at the core of synthetic biology. Automated solutions will be required to facilitate circuit design and implementation. Circuit design is increasingly being automated with design software, but innovations in experimental automation are lagging behind. Microfluidic technologies made it possible to perform in vitro transcription-translation (tx-tl) reactions with increasing throughput and sophistication, enabling screening and characterization of individual circuit elements and complete circuit designs. Here we developed an automated microfluidic cell-free processing unit (CFPU) that extends high-throughput screening capabilities to a continuous reaction environment, which is essential for the implementation and analysis of more complex and dynamic circuits. The CFPU contains 280 chemostats that can be individually programmed with DNA circuits. Each chemostat is periodically supplied with tx-tl reagents, giving rise to sustained, long-term steady state conditions. Using microfluidic pulse width modulation (PWM) the device is able to generate tx-tl reagent compositions in real-time. The device has higher throughput, lower reagent consumption, and overall higher functionality than current chemostat devices. We applied this technology to map transcription factor based repression under equilibrium conditions and implemented dynamic gene circuits switchable by small molecules. We expect the CFPU to help bridge the gap between circuit design and experimental automation for in vitro development of synthetic gene circuits.

synthetic biology↗

bioRxiv2020

Nature-inspired Circular-economy Recycling (NaCRe) for Proteins: Proof of Concept

The billion tons of synthetic polymer-based materials (i.e. plastics) produced every year are one of the greatest challenges that humanity has to face. Nature produces even more natural polymers, yet they are sustainable. For example, proteins are sequence-defined natural polymers, that are constantly recycled when living systems feed. Indeed, digestion is the protein depolymerization into amino acids (i.e. the monomers) followed by their re-assembly into new proteins of arbitrarily different sequence and function. This process breaks a common recycling paradigm where a material is recycled into itself. Organisms feed of random protein mixtures that are recycled into new proteins whose identity depends on the cells needs at the time of protein synthesis. Currently, advanced materials are increasingly made of proteins, but the abovementioned ideal recyclability of such materials has yet to be recognized and established. In this study mixtures of several (up to >30) peptides and/or proteins were depolymerized into their amino acid constituents, and these amino acids were used to synthesize new fluorescent, and bio-active proteins extra-cellularly by using an amino acid-free cell-free transcription-translation system. Proteins with high relevance in materials engineering ({beta}-lactoglobulin films, used for water filtration, or silk fibroin solutions) were successfully recycled into biotechnologically relevant proteins (green, and red fluorescent proteins, catechol 2,3-dioxygenase). The potential long-term impact of this approach to recycling lies in its compatibility with circular-economy models where raw materials remain in use as long as possible, thus reducing the burden on the planet.

biochemistry↗