An <i>E. coli</i> Cell-Free Expression Toolbox: Application to Synthetic Gene Circuits and Artificial Cells

Cell-free protein synthesis is becoming a powerful technique to construct and to study complex informational processes <i>in vitro</i>. Engineering synthetic gene circuits in a test tube, however, is seriously limited by the transcription repertoire of modern cell-free systems, composed of only a few bacteriophage regulatory elements. Here, we report the construction and the phenomenological characterization of synthetic gene circuits engineered with a cell-free expression toolbox that works with the seven <i>E. coli</i> sigma factors. The <i>E. coli</i> endogenous holoenzyme E₇₀ is used as the primary transcription machinery. Elementary circuit motifs, such as multiple stage cascades, AND gate and negative feedback loops are constructed with the six other sigma factors, two bacteriophage RNA polymerases, and a set of repressors. The circuit dynamics reveal the importance of the global mRNA turnover rate and of passive competition-induced transcriptional regulation. Cell-free reactions can be carried out over long periods of time with a small-scale dialysis reactor or in phospholipid vesicles, an artificial cell system. This toolbox is a unique platform to study complex transcription/translation-based biochemical systems <i>in vitro</i>

The All <i>E. coli</i> TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology

We report on and provide a detailed characterization of the performance and properties of a recently developed, all <i>Escherichia coli</i>, cell-free transcription and translation system. Gene expression is entirely based on the endogenous translation components and transcription machinery provided by an <i>E. coli</i> cytoplasmic extract, thus expanding the repertoire of regulatory parts to hundreds of elements. We use a powerful metabolism for ATP regeneration to achieve more than 2 mg/mL of protein synthesis in batch mode reactions, and more than 6 mg/mL in semicontinuous mode. While the strength of cell-free expression is increased by a factor of 3 on average, the output signal of simple gene circuits and the synthesis of entire bacteriophages are increased by orders of magnitude compared to previous results. Messenger RNAs and protein degradation, respectively tuned using <i>E. coli</i> MazF interferase and ClpXP AAA+ proteases, are characterized over a much wider range of rates than the first version of the cell-free toolbox. This system is a highly versatile cell-free platform to construct complex biological systems through the execution of DNA programs composed of synthetic and natural bacterial regulatory parts

Gene Circuit Performance Characterization and Resource Usage in a Cell-Free “Breadboard”

The many successes of synthetic biology have come in a manner largely different from those in other engineering disciplines; in particular, without well-characterized and simplified prototyping environments to play a role analogous to wind-tunnels in aerodynamics and breadboards in electrical engineering. However, as the complexity of synthetic circuits increases, the benefitsin cost savings and design cycle timeof a more traditional engineering approach can be significant. We have recently developed an <i>in vitro</i> “breadboard” prototyping platform based on <i>E. coli</i> cell extract that allows biocircuits to operate in an environment considerably simpler than, but functionally similar to, <i>in vivo</i>. The simplicity of this system makes it a promising tool for rapid biocircuit design and testing, as well as for probing fundamental aspects of gene circuit operation normally masked by cellular complexity. In this work, we characterize the cell-free breadboard using real-time and simultaneous measurements of transcriptional and translational activities of a small set of reporter genes and a transcriptional activation cascade. We determine the effects of promoter strength, gene concentration, and nucleoside triphosphate concentration on biocircuit properties, and we isolate the specific contributions of essential biomolecular resourcescore RNA polymerase and ribosomesto overall performance. Importantly, we show how limits on resources, particularly those involved in translation, are manifested as reduced expression in the presence of orthogonal genes that serve as additional loads on the system

Linear DNA for Rapid Prototyping of Synthetic Biological Circuits in an <i>Escherichia coli</i> Based TX-TL Cell-Free System

Accelerating the pace of synthetic biology experiments requires new approaches for rapid prototyping of circuits from individual DNA regulatory elements. However, current testing standards require days to weeks due to cloning and <i>in vivo</i> transformation. In this work, we first characterized methods to protect linear DNA strands from exonuclease degradation in an <i>Escherichia coli</i> based transcription-translation cell-free system (TX-TL), as well as mechanisms of degradation. This enabled the use of linear DNA PCR products in TX-TL. We then compared expression levels and binding dynamics of different promoters on linear DNA and plasmid DNA. We also demonstrated assembly technology to rapidly build circuits entirely <i>in vitro</i> from separate parts. Using this strategy, we prototyped a four component genetic switch in under 8 h entirely <i>in vitro.</i> Rapid <i>in vitro</i> assembly has future applications for prototyping multiple component circuits if combined with predictive computational models

Preparation of Tethered-Lipid Bilayers on Gold Surfaces for the Incorporation of Integral Membrane Proteins Synthesized by Cell-Free Expression

There is an increasing interest to express and study membrane proteins in vitro. New techniques to produce and insert functional membrane proteins into planar lipid bilayers have to be developed. In this work, we produce a tethered lipid bilayer membrane (tBLM) to provide sufficient space for the incorporation of the integral membrane protein (IMP) Aquaporin Z (AqpZ) between the tBLM and the surface of the sensor. We use a gold (Au)-coated sensor surface compatible with mechanical sensing using a quartz crystal microbalance with dissipation monitoring (QCM-D) or optical sensing using the surface plasmon resonance (SPR) method. tBLM is produced by vesicle fusion onto a thin gold film, using phospholipid-polyethylene glycol (PEG) as a spacer. Lipid vesicles are composed of 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine (POPC) and 1,2-distearoyl-<i>sn</i>-glycero-3-phosphoethanolamine-<i>N</i>-poly­(ethyleneglycol)-2000-<i>N</i>-[3-(2-pyridyldithio)­propionate], so-called DSPE-PEG-PDP, at different molar ratios (respectively, 99.5/0.5, 97.5/2.5, and 95/5 mol %), and tBLM formation is characterized using QCM-D, SPR, and atomic force technology (AFM). We demonstrate that tBLM can be produced on the gold surface after rupture of the vesicles using an α helical (AH) peptide, derived from hepatitis C virus NS5A protein, to assist the fusion process. A cell-free expression system producing the E. coli integral membrane protein Aquaporin Z (AqpZ) is directly incubated onto the tBLMs for expression and insertion of the IMP at the upper side of tBLMs. The incorporation of AqpZ into bilayers is monitored by QCM-D and compared to a control experiment (without plasmid in the cell-free expression system). We demonstrate that an IMP such as AqpZ, produced by a cell-free expression system without any protein purification, can be incorporated into an engineered tBLM preassembled at the surface of a gold-coated sensor

Semiconductor Nanoplatelets: A New Class of Ultrabright Fluorescent Probes for Cytometric and Imaging Applications

Fluorescent semiconductor nanoplatelets (NPLs) are a new generation of fluorescent probes. NPLs are colloidal two-dimensional materials that exhibit several unique optical properties, including high brightness, photostability, and extinction coefficients, as well as broad excitation and narrow emission spectra from the visible to the near-infrared spectrum. All of these exceptional fluorescence properties make NPLs interesting nanomaterials for biological applications. However, NPLs are synthesized in organic solvents and coated with hydrophobic ligands that render them insoluble in water. A current challenge is to stabilize NPLs in aqueous media compatible with biological environments. In this work, we describe a novel method to disperse fluorescent NPLs in water and functionalize them with different biomolecules for biodetection. We demonstrate that ligand exchange enables the dispersion of NPLs in water while maintaining optical properties and long-term colloidal stability in biological environments. Four different colors of NPLs were functionalized with biomolecules by random or oriented conformations. For the first time, we report that our NPLs have a higher brightness than that of standard fluorophores, like phycoerythrin or Brilliant Violet 650 (BV 650), for staining cells in flow cytometry. These results suggest that NPLs are an interesting alternative to common fluorophores for flow cytometry and imaging applications in multiplexed cellular targeting

Tuning of Recombinant Protein Expression in <i>Escherichia coli</i> by Manipulating Transcription, Translation Initiation Rates, and Incorporation of Noncanonical Amino Acids

Protein synthesis in cells has been thoroughly investigated and characterized over the past 60 years. However, some fundamental issues remain unresolved, including the reasons for genetic code redundancy and codon bias. In this study, we changed the kinetics of the <i>Eschrichia coli</i> transcription and translation processes by mutating the promoter and ribosome binding domains and by using genetic code expansion. The results expose a counterintuitive phenomenon, whereby an increase in the initiation rates of transcription and translation lead to a decrease in protein expression. This effect can be rescued by introducing slow translating codons into the beginning of the gene, by shortening gene length or by reducing initiation rates. On the basis of the results, we developed a biophysical model, which suggests that the density of co-transcriptional-translation plays a role in bacterial protein synthesis. These findings indicate how cells use codon bias to tune translation speed and protein synthesis

Mathematical Modeling of RNA-Based Architectures for Closed Loop Control of Gene Expression

Feedback allows biological systems to control gene expression precisely and reliably, even in the presence of uncertainty, by sensing and processing environmental changes. Taking inspiration from natural architectures, synthetic biologists have engineered feedback loops to tune the dynamics and improve the robustness and predictability of gene expression. However, experimental implementations of biomolecular control systems are still far from satisfying performance specifications typically achieved by electrical or mechanical control systems. To address this gap, we present mathematical models of biomolecular controllers that enable reference tracking, disturbance rejection, and tuning of the temporal response of gene expression. These controllers employ RNA transcriptional regulators to achieve closed loop control where feedback is introduced <i>via</i> molecular sequestration. Sensitivity analysis of the models allows us to identify which parameters influence the transient and steady state response of a target gene expression process, as well as which biologically plausible parameter values enable perfect reference tracking. We quantify performance using typical control theory metrics to characterize response properties and provide clear selection guidelines for practical applications. Our results indicate that RNA regulators are well-suited for building robust and precise feedback controllers for gene expression. Additionally, our approach illustrates several quantitative methods useful for assessing the performance of biomolecular feedback control systems