Study of messenger RNA inactivation and protein degradation in an Escherichia coli cell-free expression system

<p>Abstract</p> <p>Background</p> <p>A large amount of recombinant proteins can be synthesized in a few hours with <it>Escherichia coli </it>cell-free expression systems based on bacteriophage transcription. These cytoplasmic extracts are used in many applications that require large-scale protein production such as proteomics and high throughput techniques. In recent years, cell-free systems have also been used to engineer complex informational processes. These works, however, have been limited by the current available cell-free systems, which are not well adapted to these types of studies. In particular, no method has been proposed to increase the mRNA inactivation rate and the protein degradation rate in cell-free reactions. The construction of <it>in vitro </it>informational processes with interesting dynamics requires a balance between mRNA and protein synthesis (the source), and mRNA inactivation and protein degradation (the sink).</p> <p>Results</p> <p>Two quantitative studies are presented to characterize and to increase the global mRNA inactivation rate, and to accelerate the degradation of the synthesized proteins in an <it>E. coli </it>cell-free expression system driven by the endogenous RNA polymerase and sigma factor 70. The <it>E. coli </it>mRNA interferase MazF was used to increase and to adjust the mRNA inactivation rate of the <it>Firefly luciferase </it>(Luc) and of the enhanced green fluorescent protein (eGFP). Peptide tags specific to the endogenous <it>E. coli </it>AAA + proteases were used to induce and to adjust the protein degradation rate of eGFP. Messenger RNA inactivation rate, protein degradation rate, maturation time of Luc and eGFP were measured.</p> <p>Conclusions</p> <p>The global mRNA turnover and the protein degradation rate can be accelerated and tuned in a biologically relevant range in a cell-free reaction with quantitative procedures easy to implement. These features broaden the capabilities of cell-free systems with a better control of gene expression. This cell-free extract could find some applications in new research areas such as <it>in vitro </it>synthetic biology and systems biology where engineering informational processes requires a quantitative control of mRNA inactivation and protein degradation.</p

Synthetic Biology:From engineering living systems to the bottom=up construction of synthetic cells

‘What is life?’ One of the most intriguing and difficult questions to answer. Richard Feynman argued that ‘Everything that living systems do is done by atoms that act according to the laws of physics’, and this is the basis we (all) build on. Today, it is still difficult to define life, even at the cellular scale. At the molecular level, however, it is well established that life is a system of self-sustained chemical processes. Complex networks of proteins, nucleic acids and small molecules sustain the essential processes of energy provision, gene expression and cell reproduction that characterize living matter. Biochemical networks direct cell growth and division, and through the uptake of nutrients, conservation of metabolic energy and the excretion of waste, they maintain a dynamic state far from thermodynamic equilibrium. In fact, a living system that reaches equilibrium is dead

Biomolecular resource utilization in elementary cell-free gene circuits

We present a detailed dynamical model of the behavior of transcription-translation circuits in vitro that makes explicit the roles played by essential molecular resources. A set of simple two-gene test circuits operating in a cell-free biochemical 'breadboard' validate this model and highlight the consequences of limited resource availability. In particular, we are able to confirm the existence of biomolecular 'crosstalk' and isolate its individual sources. The implications of crosstalk for biomolecular circuit design and function are discussed

Linear DNA for Rapid Prototyping of Synthetic Biological Circuits in an Escherichia coli 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 in vivo transformation. In this work, we first characterized methods to protect linear DNA strands from exonuclease degradation in an Escherichia coli 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 in vitro from separate parts. Using this strategy, we prototyped a four component genetic switch in under 8 h entirely in vitro. Rapid in vitro assembly has future applications for prototyping multiple component circuits if combined with predictive computational models

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 in vitro “breadboard” prototyping platform based on E. coli cell extract that allows biocircuits to operate in an environment considerably simpler than, but functionally similar to, in vivo. 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

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform. This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology. To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems. The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted. The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters. Accompanying mathematical models are available. The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard.