http://www.jbioleng.org The latest research articles published by Journal of Biological Engineering 2010-07-21T00:00:00Z This is an RSS newsfeed from BioMed Central It is intended to be used with an RSS reader. For more information about RSS newsfeeds from BioMed Central, visit http://www.biomedcentral.com/info/about/rss/ Background: Current methods for analyzing the dynamics of natural regulatory networks, and quantifying synthetic circuit function, are limited by the lack of well-characterized genetic measurement tools. Fluorescent reporters have been used to measure dynamic gene expression, but attempts to monitor multiple genes simultaneously in single cells are relatively recent and have not focused on independent, isolated measurements. Multiple reporters can be used to observe interactions between natural genes, or to facilitate the 'debugging' of biologically engineered genetic networks. Using three distinguishable reporter genes in a single cell can reveal information not obtainable from only one or two reporters. One application of multiple reporters is the use of genetic noise to reveal regulatory connections between genes. Experiments in both natural and synthetic systems would benefit from a well-characterized platform for expressing multiple reporter genes and synthetic network components. Results: We describe such a plasmid-based platform for the design and optimization of synthetic gene networks, and for analysis of endogenous gene networks. This network scaffold consists of three distinguishable fluorescent reporter genes controlled by inducible promoters, with conveniently placed restriction sites to make modifications straightforward. We quantitatively characterize the scaffold in Escherichia coli with single-cell fluorescence imaging and time-lapse microscopy. The three spectrally distinct reporters allow independent monitoring of genetic regulation and analysis of genetic noise. As a novel application of this tool we show that the presence of genetic noise can reveal transcriptional co-regulation due to a hidden factor, and can distinguish constitutive from regulated gene expression. Conclusion: We have constructed a general chassis where three promoters from natural genes or components of synthetic networks can be easily inserted and independently monitored on a single construct using optimized fluorescent protein reporters. We have quantitatively characterized the baseline behavior of the chassis so that it can be used to measure dynamic gene regulation and noise. Overall, the system will be useful both for analyzing natural genetic networks and assembling synthetic ones. http://www.jbioleng.org/content/4/1/10 Robert Cox Mary Dunlop Michael Elowitz Journal of Biological Engineering 2010, 4:10 2010-07-21T00:00:00Z doi:10.1186/1754-1611-4-10 Journal of Biological Engineering 1754-1611 4 10 2010-07-21T00:00:00Z PDF Background: A large amount of recombinant proteins can be synthesized in a few hours with Escherichia coli 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 in vitro informational processes with interesting dynamics requires a balance between mRNA and protein synthesis (the source), and mRNA inactivation and protein degradation (the sink). Results: 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 E. coli cell-free expression system driven by the endogenous RNA polymerase and sigma factor 70. The E. coli mRNA interferase MazF was used to increase and to adjust the mRNA inactivation rate of the Firefly luciferase (Luc) and of the enhanced green fluorescent protein (eGFP). Peptide tags specific to the endogenous E. coli 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. Conclusions: 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 in vitro synthetic biology and systems biology where engineering informational processes requires a quantitative control of mRNA inactivation and protein degradation. http://www.jbioleng.org/content/4/1/9 Jonghyeon Shin Vincent Noireaux Journal of Biological Engineering 2010, 4:9 2010-07-01T00:00:00Z doi:10.1186/1754-1611-4-9 Journal of Biological Engineering 1754-1611 4 9 2010-07-01T00:00:00Z XML Background: Escherichia coli cell-free expression systems use bacteriophage RNA polymerases, such as T7, to synthesize large amounts of recombinant proteins. These systems are used for many applications in biotechnology, such as proteomics. Recently, informational processes have been reconstituted in vitro with cell-free systems. These synthetic approaches, however, have been seriously limited by a lack of transcription modularity. The current available cell-free systems have been optimized to work with bacteriophage RNA polymerases, which put significant restrictions to engineer processes related to biological information. The development of efficient cell-free systems with broader transcription capabilities is required to study complex informational processes in vitro. Results: In this work, an efficient cell-free expression system that uses the endogenous E. coli RNA polymerase only and sigma factor 70 for transcription was prepared. Approximately 0.75 mg/ml of Firefly luciferase and enhanced green fluorescent protein were produced in batch mode. A plasmid was optimized with different regulatory parts to increase the expression. In addition, a new eGFP was engineered that is more translatable in cell-free systems than the original eGFP. The protein production was characterized with three different adenosine triphosphate (ATP) regeneration systems: creatine phosphate (CP), phosphoenolpyruvate (PEP), and 3-phosphoglyceric acid (3-PGA). The maximum protein production was obtained with 3-PGA. Preparation of the crude extract was streamlined to a simple routine procedure that takes 12 hours including cell culture. Conclusions: Although it uses the endogenous E. coli transcription machinery, this cell-free system can produce active proteins in quantities comparable to bacteriophage systems. The E. coli transcription provides much more possibilities to engineer informational processes in vitro. Many E. coli promoters/operators specific to sigma factor 70 are available that form a broad library of regulatory parts. In this work, cell-free expression is developed as a toolbox to design and to study synthetic gene circuits in vitro. http://www.jbioleng.org/content/4/1/8 Jonghyeon Shin Vincent Noireaux Journal of Biological Engineering 2010, 4:8 2010-06-24T00:00:00Z doi:10.1186/1754-1611-4-8 Journal of Biological Engineering 1754-1611 4 8 2010-06-24T00:00:00Z XML The following essay was written by Mr. Alex Hatch, a junior undergraduate student majoring in Biological Engineering at Utah State University. Mr. Hatch submitted a 1000-1200 word essay to the 5th Annual Bioethics Contest sponsored by the Institute of Biological Engineering (IBE). A group of professionals in Biological Engineering assessed and ranked the essays in a blinded process. Five semi-finalists were invited to present their essays at a session at the annual meeting of IBE in Cambridge, MA on March 6, 2010. Five judges scored all the presentations and selected Mr. Hatch's contribution as the overall winner (first place). http://www.jbioleng.org/content/4/1/7 Alex Hatch Journal of Biological Engineering 2010, 4:7 2010-06-09T00:00:00Z doi:10.1186/1754-1611-4-7 Journal of Biological Engineering 1754-1611 4 7 2010-06-09T00:00:00Z XML Background: Membrane proteins are an important class of proteins, playing a key role in many biological processes, and are a promising target in pharmaceutical development. However, membrane proteins are often difficult to produce in large quantities for the purpose of crystallographic or biochemical analyses. Results: In this paper, we demonstrate that synthetic gene circuits designed specifically to overexpress certain genes can be applied to manipulate the expression kinetics of a model membrane protein, cytochrome bd quinol oxidase in E. coli, resulting in increased expression rates. The synthetic circuit involved is an engineered, autoinducer-independent variant of the lux operon activator LuxR from V. fischeri in an autoregulatory, positive feedback configuration. Conclusions: Our proof-of-concept experiments indicate a statistically significant increase in the rate of production of the bd oxidase membrane protein. Synthetic gene networks provide a feasible solution for the problem of membrane protein production. http://www.jbioleng.org/content/4/1/6 Karan Bansal Ke Yang Goutam Nistala Robert Gennis Kaustubh Bhalerao Journal of Biological Engineering 2010, 4:6 2010-05-25T00:00:00Z doi:10.1186/1754-1611-4-6 Journal of Biological Engineering 1754-1611 4 6 2010-05-25T00:00:00Z XML Background: A polarized light scattering technique was used to monitor the performance of a continuously operated foam fractionation process. The S11 and S12 parameters, elements of the light scattering matrix, combined together (S11+S12) have been correlated with the bubble size and liquid content for the case of a freely draining foam. The performance of a foam fractionation column is known to have a strong dependence on the bubble size distribution and liquid hold up in foam. In this study the enrichment is used as a metric, representative of foam properties and column performance, and correlated to the S11+S12 parameter. Results: Three different superficial gas velocities (6.9, 7.5, and 10.6 cm/min) and four different pH values (4.8, 5.5, 6.5, and 7.5) are tested for the foam fractionation of a dilute solution of bovine serum albumin (0.1 mg/ml). As a result, at scattering angle of 125° the magnitude of S11+S12 is higher as the pH increases. When the bubble sizes are small with a larger liquid content, the foam is strongly back scattering resulting in lower values of S11+S12 (at 125°) at pH = 4.8. The light scattering data and the enrichment values are measured over a period of 90 minutes and correlated using a linear model. The predictive power of the model was found to be statistically significant. Conclusion: The time average S11+S12 shows a direct proportionality with the enrichment value, indicating that polarized light should be a valuable technique for monitoring foam fractionation columns. Additional knowledge of the nature of dependence between foam properties and S11+S12 combined with models relating the enrichment to the bubble size and liquid hold up is needed to develop an accurate diagnostics tool for monitoring enrichment utilizing S11+S12 measurements. http://www.jbioleng.org/content/4/1/5 Janakiraman Swamy Czarena Crofcheck M Menguc Journal of Biological Engineering 2010, 4:5 2010-04-15T00:00:00Z doi:10.1186/1754-1611-4-5 Journal of Biological Engineering 1754-1611 4 5 2010-04-15T00:00:00Z XML Background: Positive feedback is a common mechanism used in the regulation of many gene circuits as it can amplify the response to inducers and also generate binary outputs and hysteresis. In the context of electrical circuit design, positive feedback is often considered in the design of amplifiers. Similar approaches, therefore, may be used for the design of amplifiers in synthetic gene circuits with applications, for example, in cell-based sensors. Results: We developed a modular positive feedback circuit that can function as a genetic signal amplifier, heightening the sensitivity to inducer signals as well as increasing maximum expression levels without the need for an external cofactor. The design utilizes a constitutively active, autoinducer-independent variant of the quorum-sensing regulator LuxR. We experimentally tested the ability of the positive feedback module to separately amplify the output of a one-component tetracycline sensor and a two-component aspartate sensor. In each case, the positive feedback module amplified the response to the respective inducers, both with regards to the dynamic range and sensitivity. Conclusions: The advantage of our design is that the actual feedback mechanism depends only on a single gene and does not require any other modulation. Furthermore, this circuit can amplify any transcriptional signal, not just one encoded within the circuit or tuned by an external inducer. As our design is modular, it can potentially be used as a component in the design of more complex synthetic gene circuits. http://www.jbioleng.org/content/4/1/4 Goutam Nistala Kang Wu Christopher Rao Kaustubh Bhalerao Journal of Biological Engineering 2010, 4:4 2010-02-26T00:00:00Z doi:10.1186/1754-1611-4-4 Journal of Biological Engineering 1754-1611 4 4 2010-02-26T00:00:00Z XML Background: The engineering of metabolism holds tremendous promise for the production of desirable metabolites, particularly alternative fuels and other highly reduced molecules. Engineering approaches must redirect the transfer of chemical reducing equivalents, preventing these electrons from being lost to general cellular metabolism. This is especially the case for high energy electrons stored in iron-sulfur clusters within proteins, which are readily transferred when two such clusters are brought in close proximity. Iron sulfur proteins therefore require mechanisms to ensure interaction between proper partners, analogous to many signal transduction proteins. While there has been progress in the isolation of engineered metabolic pathways in recent years, the design of insulated electron metabolism circuits in vivo has not been pursued. Results: Here we show that a synthetic hydrogen-producing electron transfer circuit in Escherichia coli can be insulated from existing cellular metabolism via multiple approaches, in many cases improving the function of the pathway. Our circuit is composed of heterologously expressed [Fe-Fe]-hydrogenase, ferredoxin, and pyruvate-ferredoxin oxidoreductase (PFOR), allowing the production of hydrogen gas to be coupled to the breakdown of glucose. We show that this synthetic pathway can be insulated through the deletion of competing reactions, rational engineering of protein interaction surfaces, direct protein fusion of interacting partners, and co-localization of pathway components on heterologous protein scaffolds. Conclusions: Through the construction and characterization of a synthetic metabolic circuit in vivo, we demonstrate a novel system that allows for predictable engineering of an insulated electron transfer pathway. The development of this system demonstrates working principles for the optimization of engineered pathways for alternative energy production, as well as for understanding how electron transfer between proteins is controlled. http://www.jbioleng.org/content/4/1/3 Christina Agapakis Daniel Ducat Patrick Boyle Edwin Wintermute Jeffrey Way Pamela Silver Journal of Biological Engineering 2010, 4:3 2010-02-25T00:00:00Z doi:10.1186/1754-1611-4-3 Journal of Biological Engineering 1754-1611 4 3 2010-02-25T00:00:00Z XML Background: The highly reactive aldehyde acrolein is a very potent endogenous toxin with a long half-life. Acrolein is produced within cells after insult, and is a central player in slow and progressive "secondary injury" cascades. Indeed, acrolein-biomolecule complexes formed by cross-linking with proteins and DNA are associated with a number of pathologies, especially central nervous system (CNS) trauma and neurodegenerative diseases. Hydralazine is capable of inhibiting or reducing acrolein-induced damage. However, since hydralazine's principle activity is to reduce blood pressure as a common anti-hypertension drug, the possible problems encountered when applied to hypotensive trauma victims have led us to explore alternative approaches. This study aims to evaluate such an alternative - a chitosan nanoparticle-based therapeutic system. Results: Hydralazine-loaded chitosan nanoparticles were prepared using different types of polyanions and characterized for particle size, morphology, zeta potential value, and the efficiency of hydralazine entrapment and release. Hydralazine-loaded chitosan nanoparticles ranged in size from 300 nm to 350 nm in diameter, and with a tunable, or adjustable, surface charge. Conclusions: We evaluated the utility of chitosan nanoparticles with an in-vitro model of acrolein-mediated cell injury using PC -12 cells. The particles effectively, and statistically, reduced damage to membrane integrity, secondary oxidative stress, and lipid peroxidation. This study suggests that a chitosan nanoparticle-based therapy to interfere with "secondary" injury may be possible. http://www.jbioleng.org/content/4/1/2 Youngnam Cho Riyi Shi Richard Borgens Journal of Biological Engineering 2010, 4:2 2010-01-29T00:00:00Z doi:10.1186/1754-1611-4-2 Journal of Biological Engineering 1754-1611 4 2 2010-01-29T00:00:00Z XML Background: Standard biological parts, such as BioBricks™ parts, provide the foundation for a new engineering discipline that enables the design and construction of synthetic biological systems with a variety of applications in bioenergy, new materials, therapeutics, and environmental remediation. Although the original BioBricks™ assembly standard has found widespread use, it has several shortcomings that limit its range of potential applications. In particular, the system is not suitable for the construction of protein fusions due to an unfavorable scar sequence that encodes an in-frame stop codon. Results: Here, we present a similar but new composition standard, called BglBricks, that addresses the scar translation issue associated with the original standard. The new system employs BglII and BamHI restriction enzymes, robust cutters with an extensive history of use, and results in a 6-nucleotide scar sequence encoding glycine-serine, an innocuous peptide linker in most protein fusion applications. We demonstrate the utility of the new standard in three distinct applications, including the construction of constitutively active gene expression devices with a wide range of expression profiles, the construction of chimeric, multi-domain protein fusions, and the targeted integration of functional DNA sequences into specific loci of the E. coli genome. Conclusions: The BglBrick standard provides a new, more flexible platform from which to generate standard biological parts and automate DNA assembly. Work on BglBrick assembly reactions, as well as on the development of automation and bioinformatics tools, is currently underway. These tools will provide a foundation from which to transform genetic engineering from a technically intensive art into a purely design-based discipline. http://www.jbioleng.org/content/4/1/1 J Anderson John Dueber Mariana Leguia Gabriel Wu Jonathan Goler Adam Arkin Jay Keasling Journal of Biological Engineering 2010, 4:1 2010-01-20T00:00:00Z doi:10.1186/1754-1611-4-1 Journal of Biological Engineering 1754-1611 4 1 2010-01-20T00:00:00Z XML