Iterative design of training data to control intricate enzymatic reaction networks.

Kinetic modeling of in vitro enzymatic reaction networks is vital to understand and control the complex behaviors emerging from the nonlinear interactions inside. However, modeling is severely hampered by the lack of training data. Here, we introduce a methodology that combines an active learning-like approach and flow chemistry to efficiently create optimized datasets for a highly interconnected enzymatic reactions network with multiple sub-pathways. The optimal experimental design (OED) algorithm designs a sequence of out-of-equilibrium perturbations to maximize the information about the reaction kinetics, yielding a descriptive model that allows control of the output of the network towards any cost function. We experimentally validate the model by forcing the network to produce different product ratios while maintaining a minimum level of overall conversion efficiency. Our workflow scales with the complexity of the system and enables the optimization of previously unobtainable network outputs.

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 >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.

Cell-Free Expression System Derived from a Near-Minimal Synthetic Bacterium.

Cell-free expression (CFE) systems are fundamental to reconstituting metabolic pathways in vitro toward the construction of a synthetic cell. Although an Escherichia coli-based CFE system is well-established, simpler model organisms are necessary to understand the principles behind life-like behavior. Here, we report the successful creation of a CFE system derived from JCVI-syn3A (Syn3A), the minimal synthetic bacterium. Previously, high ribonuclease activity in Syn3A lysates impeded the establishment of functional CFE systems. Now, we describe how an unusual cell lysis method (nitrogen decompression) yielded Syn3A lysates with reduced ribonuclease activity that supported in vitro expression. To improve the protein yields in the Syn3A CFE system, we optimized the Syn3A CFE reaction mixture using an active machine learning tool. The optimized reaction mixture improved the CFE 3.2-fold compared to the preoptimized condition. This is the first report of a functional CFE system derived from a minimal synthetic bacterium, enabling further advances in bottom-up synthetic biology.

Traditional protocols and optimization methods lead to absent expression in a mycoplasma cell-free gene expression platform.

Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a Mycoplasma bacterium-based CFE system using lysates of the genome-minimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative Mycoplasma capricolum (Mcap). To produce mycoplasma-derived crude lysates, we systematically tested methods commonly used for bacteria, based on the S30 protocol of Escherichia coli. Unexpectedly, after numerous attempts to optimize lysate production methods or composition of feeding buffer, none of the Mcap or Syn3A lysates supported cell-free gene expression. Only modest levels of in vitro transcription of RNA aptamers were observed. While our experimental systems were intended to perform transcription and translation, our assays focused on RNA. Further investigations identified persistently high ribonuclease (RNase) activity in all lysates, despite removal of recognizable nucleases from the respective genomes and attempts to inhibit nuclease activities in assorted CFE preparations. An alternative method using digitonin to permeabilize the mycoplasma cell membrane produced a lysate with diminished RNase activity yet still was unable to support cell-free gene expression. We found that intact mycoplasma cells poisoned E. coli cell-free extracts by degrading ribosomal RNAs, indicating that the mycoplasma cells, even the minimal cell, have a surface-associated RNase activity. However, it is not clear which gene encodes the RNase. This work summarizes attempts to produce mycoplasma-based CFE and serves as a cautionary tale for researchers entering this field. Graphical Abstract.

Improving Breast Cancer Treatment Specificity Using Aptamers Obtained by 3D Cell-SELEX.

Three-dimensional spheroids of non-malignant MCF10A and malignant SKBR3 breast cells were used for subsequent 3D Cell-SELEX to generate aptamers for specific binding and treatment of breast cancer cells. Using 3D Cell-SELEX combined with Next-Generation Sequencing and bioinformatics, ten abundant aptamer families with specific structures were identified that selectively bind to SKBR3, and not to MCF10A cells. Multivalent aptamer polymers were synthesized by co-polymerization and analyzed for binding performance as well as therapeutic efficacy. Binding performance was determined by confocal fluorescence imaging and revealed specific binding and efficient internalization of aptamer polymers into SKBR3 spheroids. For therapeutic purposes, DNA sequences that intercalate the cytotoxic drug doxorubicin were co-polymerized into the aptamer polymers. Viability tests show that the drug-loaded polymers are specific and effective in killing SKBR3 breast cancer cells. Thus, the 3D-selected aptamers enhanced the specificity of doxorubicin against malignant over non-malignant breast cells. The innovative modular DNA aptamer platform based on 3D Cell SELEX and polymer multivalency holds great promise for diagnostics and treatment of breast cancer.

Multivalent Sgc8c-aptamer decorated polymer scaffolds for leukemia targeting.

T-cell acute lymphoblastic leukemia causes a disproportional amount of immature white blood cells in the patients' bone marrow. The significant undesired side effects associated with traditional chemotherapy treatment prompted us to study a more effective treatment strategy. We decorated polyisocyanopeptide scaffolds with the selective leukemia cell binding aptamer sgc8c and found that the polymers inhibit proliferation by G0/G1-phase arrest, serving as an opportunity for future therapeutic strategies.

Transcription and Translation in Cytomimetic Protocells Perform Most Efficiently at Distinct Macromolecular Crowding Conditions.

The formation of cytomimetic protocells that capture the physicochemical aspects of living cells is an important goal in bottom-up synthetic biology. Here, we recreated the crowded cytoplasm in liposome-based protocells and studied the kinetics of cell-free gene expression in these crowded containers. We found that diffusion of key components is affected not only by macromolecular crowding but also by enzymatic activity in the protocell. Surprisingly, size-dependent diffusion in crowded conditions yielded two distinct maxima for protein synthesis, reflecting the differential impact of crowding on transcription and translation. Our experimental data show, for the first time, that macromolecular crowding induces a switch from reaction to diffusion control and that this switch depends on the sizes of the macromolecules involved. These results highlight the need to control the physical environment in the design of synthetic cells.

Ramified rolling circle amplification for synthesis of nucleosomal DNA sequences.

Nucleosomes are a crucial platform for the recruitment and assembly of protein complexes that process the DNA. Mechanistic and structural in vitro studies typically rely on recombinant nucleosomes that are reconstituted using artificial, strong-positioning DNA sequences. To facilitate such studies on native, genomic nucleosomes, there is a need for methods to produce any desired DNA sequence in an efficient manner. The current methods either do not offer much flexibility in choice of sequence or are less efficient in yield and labor. Here, we show that ramified rolling circle amplification (RCA) can be used to produce milligram amounts of a genomic nucleosomal DNA fragment in a scalable, one-pot reaction overnight. The protocol is efficient and flexible in choice of DNA sequence. It yields 10-fold more product than PCR, and rivals production using plasmids. We demonstrate the approach by producing the genomic DNA from the human LIN28B locus and show that it forms functional nucleosomes capable of binding pioneer transcription factor Oct4.

Dissipative adaptation in driven self-assembly leading to self-dividing fibrils.

Out-of-equilibrium self-assembly of proteins such as actin and tubulin is a key regulatory process controlling cell shape, motion and division. The design of functional nanosystems based on dissipative self-assembly has proven to be remarkably difficult due to a complete lack of control over the spatial and temporal characteristics of the assembly process. Here, we show the dissipative self-assembly of FtsZ protein (a bacterial homologue of tubulin) within coacervate droplets. More specifically, we show how such barrier-free compartments govern the local availability of the energy-rich building block guanosine triphosphate, yielding highly dynamic fibrils. The increased flux of FtsZ monomers at the tips of the fibrils results in localized FtsZ assembly, elongation of the coacervate compartments, followed by division of the fibrils into two. We rationalize the directional growth and division of the fibrils using dissipative reaction-diffusion kinetics and capillary action of the filaments as main inputs. The principle presented here, in which open compartments are used to modulate the rates of dissipative self-assembly by restricting the absorption of energy from the environment, may provide a general route to dissipatively adapting nanosystems exhibiting life-like behaviour.

Biomimetic Stress Sensitive Hydrogel Controlled by DNA Nanoswitches.

One of the most intriguing and important aspects of biological supramolecular materials is its ability to adapt macroscopic properties in response to environmental cues for controlling cellular processes. Recently, bulk matrix stiffness, in particular, stress sensitivity, has been established as a key mechanical cue in cellular function and development. However, stress-stiffening capacity and the ability to control and exploit this key characteristic is relatively new to the field of biomimetic materials. In this work, DNA-responsive hydrogels, composed of semiflexible PIC polymers equipped with DNA cross-linkers, were engineered to create mimics of natural biopolymer networks that capture these essential elastic properties and can be controlled by external stimuli. We show that the elastic properties are governed by the molecular structure of the cross-linker, which can be readily varied providing access to a broad range of highly tunable soft hydrogels with diverse stress-stiffening regimes. By using cross-linkers based on DNA nanoswitches, responsive to pH or ligands, internal control elements of mechanical properties are implemented that allow for dynamic control of elastic properties with high specificity. The work broadens the current knowledge necessary for the development of user defined biomimetic materials with stress stiffening capacity.

A membrane-anchored aptamer sensor for probing IFNγ secretion by single cells.

Insight into the behavior of individual immune cells, in particular cytokine secretion, will contribute to a more fundamental understanding of the immune system. In this work, we have developed a cell membrane-anchored sensor for the detection of cytokines secreted by single cells using a combination of aptamer-based sensors and droplet microfluidics.

Stable isotope labeling methods for DNA.

NMR is a powerful method for studying proteins and nucleic acids in solution. The study of nucleic acids by NMR is far more challenging than for proteins, which is mainly due to the limited number of building blocks and unfavorable spectral properties. For NMR studies of DNA molecules, (site specific) isotope enrichment is required to facilitate specific NMR experiments and applications. Here, we provide a comprehensive review of isotope-labeling strategies for obtaining stable isotope labeled DNA as well as specifically stable isotope labeled building blocks required for enzymatic DNA synthesis.

Protein Synthesis in Coupled and Uncoupled Cell-Free Prokaryotic Gene Expression Systems.

Secondary structure formation of mRNA, caused by desynchronization of transcription and translation, is known to impact gene expression in vivo. Yet, inactivation of mRNA by secondary structures in cell-free protein expression is frequently overlooked. Transcription and translation rates are often not highly synchronized in cell-free expression systems, leading to a temporal mismatch between the processes and a drop in efficiency of protein production. By devising a cell-free gene expression platform in which transcriptional and translational elongation are successfully performed independently, we determine that sequence-dependent mRNA secondary structures are the main cause of mRNA inactivation in in vitro gene expression.

Macromolecular crowding creates heterogeneous environments of gene expression in picolitre droplets.

Understanding the dynamics of complex enzymatic reactions in highly crowded small volumes is crucial for the development of synthetic minimal cells. Compartmentalized biochemical reactions in cell-sized containers exhibit a degree of randomness due to the small number of molecules involved. However, it is unknown how the physical environment contributes to the stochastic nature of multistep enzymatic processes. Here, we present a robust method to quantify gene expression noise in vitro using droplet microfluidics. We study the changes in stochasticity in the cell-free gene expression of two genes compartmentalized within droplets as a function of DNA copy number and macromolecular crowding. We find that decreased diffusion caused by a crowded environment leads to the spontaneous formation of heterogeneous microenvironments of mRNA as local production rates exceed the diffusion rates of macromolecules. This heterogeneity leads to a higher probability of the molecular machinery staying in the same microenvironment, directly increasing the system's stochasticity.

Cell-Like Nanostructured Environments Alter Diffusion and Reaction Kinetics in Cell-Free Gene Expression.

In highly crowded and viscous intracellular environments, the kinetics of complex enzymatic reactions are determined by both reaction and diffusion rates. However in vitro studies on transcription and translation often fail to take into account the density of the prokaryotic cytoplasm. Here we mimic the cellular environment by using a porous hydrogel matrix, to study the effects of macromolecular crowding on gene expression. We found that within microgels gene expression is localized, transcription is enhanced up to fivefold, and translation is enhanced up to fourfold. Our results highlight the need to consider the role of the physical environment on complex biochemical reactions, in this case macromolecular crowding, nanoscale spatial organization, and confinement.

Associative Interactions in Crowded Solutions of Biopolymers Counteract Depletion Effects.

The cytosol of Escherichia coli is an extremely crowded environment, containing high concentrations of biopolymers which occupy 20-30% of the available volume. Such conditions are expected to yield depletion forces, which strongly promote macromolecular complexation. However, crowded macromolecule solutions, like the cytosol, are very prone to nonspecific associative interactions that can potentially counteract depletion. It remains unclear how the cytosol balances these opposing interactions. We used a FRET-based probe to systematically study depletion in vitro in different crowded environments, including a cytosolic mimic, E. coli lysate. We also studied bundle formation of FtsZ protofilaments under identical crowded conditions as a probe for depletion interactions at much larger overlap volumes of the probe molecule. The FRET probe showed a more compact conformation in synthetic crowding agents, suggesting strong depletion interactions. However, depletion was completely negated in cell lysate and other protein crowding agents, where the FRET probe even occupied slightly more volume. In contrast, bundle formation of FtsZ protofilaments proceeded as readily in E. coli lysate and other protein solutions as in synthetic crowding agents. Our experimental results and model suggest that, in crowded biopolymer solutions, associative interactions counterbalance depletion forces for small macromolecules. Furthermore, the net effects of macromolecular crowding will be dependent on both the size of the macromolecule and its associative interactions with the crowded background.

Production of Homogeneous Recombinant RNA Using a tRNA Scaffold and Hammerhead Ribozymes.

Bacterial overproduction of recombinant RNA using a tRNA scaffold yields large amounts of chimeric RNA. For structural and functional characterizations of the RNA it is often necessary to remove the scaffold. Here we describe an efficient and facile method to release the RNA of interest from the tRNA scaffold by selective cleavage using cis-acting hammerhead ribozymes. After cleavage, the RNA of interest is purified to homogeneity using standard chromatographic and electrophoretic methods. Up to 5 mg of highly pure end-product RNA can be obtained from a single liter of bacterial culture.

Enzymatic preparation of multimilligram amounts of pure single-stranded DNA samples for material and analytical sciences.

We present a method for high-yield production of multimilligram amounts of pure single-stranded DNA employing rolling circle amplification (RCA) and processing by restriction enzymes. Pure and homogeneous samples are produced with minimal handling time, reagents, and waste products. The RCA method is more than twice as efficient in dNTP incorporation than conventional polymerase chain reaction in producing end product. The validity and utility of the method are demonstrated in the production of a uniformly (13)C/(15)N-labeled 38-nt cocaine aptamer DNA used in nanosensing devices.

Structural and thermodynamic signatures that define pseudotriloop RNA hairpins.

Pseudotriloop (PTL) structures in RNAs have been recognized as essential elements in RNA folding and recognition of proteins. PTL structures are derived from hexaloops by formation of a cross-loop base pair leaving a triloop and 3' bulged out residue. Despite their common presence and functional importance, insufficient structural and thermodynamic data are available that can be used to predict formation of PTLs from sequence alone. Using NMR spectroscopy and UV-melting data we established factors that contribute to the formation and stability of PTL structures derived from hepatitis B virus and human foamy virus. The NMR data show that, besides the cross-loop base pair, also a 3' pyrimidine bulge and a G-C loop-closing base pair are primary determinants of PTL formation. By changing the G-C closing base pair into C-G, the PTL switches into a hexaloop. Comparison of these rules with regular triloop hairpins and PTLs from other sources is discussed as well as the conservation of a PTL in human foamy virus and other spumaretroviruses.

Enhanced transcription rates in membrane-free protocells formed by coacervation of cell lysate.

Liquid-liquid phase transitions in complex mixtures of proteins and other molecules produce crowded compartments supporting in vitro transcription and translation. We developed a method based on picoliter water-in-oil droplets to induce coacervation in Escherichia coli cell lysate and follow gene expression under crowded and noncrowded conditions. Coacervation creates an artificial cell-like environment in which the rate of mRNA production is increased significantly. Fits to the measured transcription rates show a two orders of magnitude larger binding constant between DNA and T7 RNA polymerase, and five to six times larger rate constant for transcription in crowded environments, strikingly similar to in vivo rates. The effect of crowding on interactions and kinetics of the fundamental machinery of gene expression has a direct impact on our understanding of biochemical networks in vivo. Moreover, our results show the intrinsic potential of cellular components to facilitate macromolecular organization into membrane-free compartments by phase separation.