http://www.jbioleng.org The most accessed 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: 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: 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: The underlying goal of synthetic biology is to make the process of engineering biological systems easier. Recent work has focused on defining and developing standard biological parts. The technical standard that has gained the most traction in the synthetic biology community is the BioBrick standard for physical composition of genetic parts. Parts that conform to the BioBrick assembly standard are BioBrick standard biological parts. To date, over 2,000 BioBrick parts have been contributed to, and are available from, the Registry of Standard Biological Parts. Results: Here we extended the same advantages of BioBrick standard biological parts to the plasmid-based vectors that are used to provide and propagate BioBrick parts. We developed a process for engineering BioBrick vectors from BioBrick parts. We designed a new set of BioBrick parts that encode many useful vector functions. We combined the new parts to make a BioBrick base vector that facilitates BioBrick vector construction. We demonstrated the utility of the process by constructing seven new BioBrick vectors. We also successfully used the resulting vectors to assemble and propagate other BioBrick standard biological parts. Conclusion: We extended the principles of part reuse and standardization to BioBrick vectors. As a result, myriad new BioBrick vectors can be readily produced from all existing and newly designed BioBrick parts. We invite the synthetic biology community to (1) use the process to make and share new BioBrick vectors; (2) expand the current collection of BioBrick vector parts; and (3) characterize and improve the available collection of BioBrick vector parts. http://www.jbioleng.org/content/2/1/5 Reshma Shetty Drew Endy Thomas Knight Journal of Biological Engineering 2008, 2:5 2008-04-14T00:00:00Z doi:10.1186/1754-1611-2-5 Journal of Biological Engineering 1754-1611 2 5 2008-04-14T00:00:00Z XML Background: The engineering of many-component, synthetic biological systems is being made easier by the development of collections of reusable, standard biological parts. However, the complexity of biology makes it difficult to predict the extent to which such efforts will succeed. As a first practical example, the Registry of Standard Biological Parts started at MIT now maintains and distributes thousands of BioBrick™ standard biological parts. However, BioBrick parts are only standardized in terms of how individual parts are physically assembled into multi-component systems, and most parts remain uncharacterized. Standardized tools, techniques, and units of measurement are needed to facilitate the characterization and reuse of parts by independent researchers across many laboratories. Results: We found that the absolute activity of BioBrick promoters varies across experimental conditions and measurement instruments. We choose one promoter (BBa_J23101) to serve as an in vivo reference standard for promoter activity. We demonstrated that, by measuring the activity of promoters relative to BBa_J23101, we could reduce variation in reported promoter activity due to differences in test conditions and measurement instruments by ~50%. We defined a Relative Promoter Unit (RPU) in order to report promoter characterization data in compatible units and developed a measurement kit so that researchers might more easily adopt RPU as a standard unit for reporting promoter activity. We distributed a set of test promoters to multiple labs and found good agreement in the reported relative activities of promoters so measured. We also characterized the relative activities of a reference collection of BioBrick promoters in order to further support adoption of RPU-based measurement standards. Conclusion: Relative activity measurements based on an in vivoreference standard enables improved measurement of promoter activity given variation in measurement conditions and instruments. These improvements are sufficient to begin to support the measurement of promoter activities across many laboratories. Additional in vivo reference standards for other types of biological functions would seem likely to have similar utility, and could thus improve research on the design, production, and reuse of standard biological parts. http://www.jbioleng.org/content/3/1/4 Jason Kelly Adam Rubin Joseph Davis Caroline Ajo-Franklin John Cumbers Michael Czar Kim de Mora Aaron Glieberman Dileep Monie Drew Endy Journal of Biological Engineering 2009, 3:4 2009-03-20T00:00:00Z doi:10.1186/1754-1611-3-4 Journal of Biological Engineering 1754-1611 3 4 2009-03-20T00:00:00Z XML Background: The Hamiltonian Path Problem asks whether there is a route in a directed graph from a beginning node to an ending node, visiting each node exactly once. The Hamiltonian Path Problem is NP complete, achieving surprising computational complexity with modest increases in size. This challenge has inspired researchers to broaden the definition of a computer. DNA computers have been developed that solve NP complete problems. Bacterial computers can be programmed by constructing genetic circuits to execute an algorithm that is responsive to the environment and whose result can be observed. Each bacterium can examine a solution to a mathematical problem and billions of them can explore billions of possible solutions. Bacterial computers can be automated, made responsive to selection, and reproduce themselves so that more processing capacity is applied to problems over time. Results: We programmed bacteria with a genetic circuit that enables them to evaluate all possible paths in a directed graph in order to find a Hamiltonian path. We encoded a three node directed graph as DNA segments that were autonomously shuffled randomly inside bacteria by a Hin/hixC recombination system we previously adapted from Salmonella typhimurium for use in Escherichia coli. We represented nodes in the graph as linked halves of two different genes encoding red or green fluorescent proteins. Bacterial populations displayed phenotypes that reflected random ordering of edges in the graph. Individual bacterial clones that found a Hamiltonian path reported their success by fluorescing both red and green, resulting in yellow colonies. We used DNA sequencing to verify that the yellow phenotype resulted from genotypes that represented Hamiltonian path solutions, demonstrating that our bacterial computer functioned as expected. Conclusion: We successfully designed, constructed, and tested a bacterial computer capable of finding a Hamiltonian path in a three node directed graph. This proof-of-concept experiment demonstrates that bacterial computing is a new way to address NP-complete problems using the inherent advantages of genetic systems. The results of our experiments also validate synthetic biology as a valuable approach to biological engineering. We designed and constructed basic parts, devices, and systems using synthetic biology principles of standardization and abstraction. http://www.jbioleng.org/content/3/1/11 Jordan Baumgardner Karen Acker Oyinade Adefuye Samuel Crowley Will DeLoache James Dickson Lane Heard Andrew Martens Nickolaus Morton Michelle Ritter Amber Shoecraft Jessica Treece Matthew Unzicker Amanda Valencia Mike Waters A. Campbell Laurie Heyer Jeffrey Poet Todd Eckdahl Journal of Biological Engineering 2009, 3:11 2009-07-24T00:00:00Z doi:10.1186/1754-1611-3-11 Journal of Biological Engineering 1754-1611 3 11 2009-07-24T00:00:00Z 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 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: We investigated the possibility of executing DNA-based computation in living cells by engineering Escherichia coli to address a classic mathematical puzzle called the Burnt Pancake Problem (BPP). The BPP is solved by sorting a stack of distinct objects (pancakes) into proper order and orientation using the minimum number of manipulations. Each manipulation reverses the order and orientation of one or more adjacent objects in the stack. We have designed a system that uses site-specific DNA recombination to mediate inversions of genetic elements that represent pancakes within plasmid DNA. Results: Inversions (or "flips") of the DNA fragment pancakes are driven by the Salmonella typhimurium Hin/hix DNA recombinase system that we reconstituted as a collection of modular genetic elements for use in E. coli. Our system sorts DNA segments by inversions to produce different permutations of a promoter and a tetracycline resistance coding region; E. coli cells become antibiotic resistant when the segments are properly sorted. Hin recombinase can mediate all possible inversion operations on adjacent flippable DNA fragments. Mathematical modeling predicts that the system reaches equilibrium after very few flips, where equal numbers of permutations are randomly sorted and unsorted. Semiquantitative PCR analysis of in vivo flipping suggests that inversion products accumulate on a time scale of hours or days rather than minutes. Conclusion: The Hin/hix system is a proof-of-concept demonstration of in vivo computation with the potential to be scaled up to accommodate larger and more challenging problems. Hin/hix may provide a flexible new tool for manipulating transgenic DNA in vivo. http://www.jbioleng.org/content/2/1/8 Karmella Haynes Marian Broderick Adam Brown Trevor Butner James Dickson Lance Harden Lane Heard Eric Jessen Kelly Malloy Brad Ogden Sabriya Rosemond Samantha Simpson Erin Zwack Malcolm Campbell Todd Eckdahl Laurie Heyer Jeffrey Poet Journal of Biological Engineering 2008, 2:8 2008-05-20T00:00:00Z doi:10.1186/1754-1611-2-8 Journal of Biological Engineering 1754-1611 2 8 2008-05-20T00:00:00Z XML Background: The release of heavy metal-containing nanoparticles (NP) into the environment may be harmful to the efficacy of beneficial microbes that function in element cycling, pollutant degradation and plant growth. Nanoparticles of Ag, CuO and ZnO are of interest as antimicrobials against pathogenic bacteria. We demonstrate here their antimicrobial activity against the beneficial soil microbe, Pseudomonas putida KT2440. Results: Toxicity was detected in a KT2440 construct possessing a plasmid bearing the luxAB reporter genes. "As manufactured" preparations of nano- Ag, -CuO and -ZnO caused rapid dose-dependent loss of light output in the biosensor. Cell death accompanied loss in Lux activity with treatments by nano-Ag and -CuO, but with -ZnO the treatments were bacteriostatic rather than bactericidal. Bulk equivalents of these products showed no inhibitory activity, indicating that particle size was determinant in activity. Flow Field-Flow Fractionation (FlFFF) of an aqueous suspension of the nano-CuO and ZnO revealed a small proportion of 5 nm NP and aggregated particulates with sizes ranging between 70 nm and 300 nm; the majority portion of material was aggregated into particles larger than 300 nm in size. Thus within the commercial preparation there may be microbially active and inactive forms. Conclusion: The "as-made" NP of Ag, CuO and ZnO have toxic effects on a beneficial soil microbe, leading to bactericidal or bacteriostatic effects depending on the NP employed. The lack of toxicity from bulk materials suggests that aggregation of the NP into larger particles, possibly by factors present in the environment may reduce their nontarget antimicrobial activity. http://www.jbioleng.org/content/3/1/9 Priyanka Gajjar Brian Pettee David Britt Wenjie Huang William Johnson Anne Anderson Journal of Biological Engineering 2009, 3:9 2009-06-26T00:00:00Z doi:10.1186/1754-1611-3-9 Journal of Biological Engineering 1754-1611 3 9 2009-06-26T00:00:00Z XML Background: Synthetic biology brings together concepts and techniques from engineering and biology. In this field, computer-aided design (CAD) is necessary in order to bridge the gap between computational modeling and biological data. Using a CAD application, it would be possible to construct models using available biological "parts" and directly generate the DNA sequence that represents the model, thus increasing the efficiency of design and construction of synthetic networks. Results: An application named TinkerCell has been developed in order to serve as a CAD tool for synthetic biology. TinkerCell is a visual modeling tool that supports a hierarchy of biological parts. Each part in this hierarchy consists of a set of attributes that define the part, such as sequence or rate constants. Models that are constructed using these parts can be analyzed using various third-party C and Python programs that are hosted by TinkerCell via an extensive C and Python application programming interface (API). TinkerCell supports the notion of a module, which are networks with interfaces. Such modules can be connected to each other, forming larger modular networks. TinkerCell is a free and open-source project under the Berkeley Software Distribution license. Downloads, documentation, and tutorials are available at http://www.tinkercell.com. Conclusion: An ideal CAD application for engineering biological systems would provide features such as: building and simulating networks, analyzing robustness of networks, and searching databases for components that meet the design criteria. At the current state of synthetic biology, there are no established methods for measuring robustness or identifying components that fit a design. The same is true for databases of biological parts. TinkerCell's flexible modeling framework allows it to cope with changes in the field. Such changes may involve the way parts are characterized or the way synthetic networks are modeled and analyzed computationally. TinkerCell can readily accept third-party algorithms, allowing it to serve as a platform for testing different methods relevant to synthetic biology. http://www.jbioleng.org/content/3/1/19 Deepak Chandran Frank Bergmann Herbert Sauro Journal of Biological Engineering 2009, 3:19 2009-10-29T00:00:00Z doi:10.1186/1754-1611-3-19 Journal of Biological Engineering 1754-1611 3 19 2009-10-29T00:00:00Z XML