http://www.jbioleng.org The most accessed research articles published by Journal of Biological Engineering 2012-04-25T00:00:00Z 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, null:11 2009-07-24T00:00:00Z doi:10.1186/1754-1611-3-11 Bacterial computer solves complex math problem E. coli cells can be engineered to evaluate possible combinations of expressed genes and determine which is correct given a predetermined outcome, similar to the mathematical Hamiltonian path problem, providing proof of concept for bacterial computers solving other NP-complete problems. /content/figures/1754-1611-3-11-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 11 2009-07-24T00:00:00Z 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, null:8 2008-05-20T00:00:00Z doi:10.1186/1754-1611-2-8 Genetically altered bacteria create "living computers" E. coli cells can be engineered to sort DNA fragments into a specific order and orientation, similar to the mathematical burnt pancake problem, providing a flexible new tool for manipulating transgenic DNA in vivo. /content/figures/1754-1611-2-8-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 8 2008-05-20T00:00:00Z XML Background: Recombinant protein production is a process of great industrial interest, with products that range from pharmaceuticals to biofuels. Since high level production of recombinant protein imposes significant stress in the host organism, several methods have been developed over the years to optimize protein production. So far, these trial-and-error techniques have proved laborious and sensitive to process parameters, while there has been no attempt to address the problem by applying Synthetic Biology principles and methods, such as integration of standardized parts in novel synthetic circuits. Results: We present a novel self-regulatory protein production system that couples the control of recombinant protein production with a stress-induced, negative feedback mechanism. The synthetic circuit allows the down-regulation of recombinant protein expression through a stress-induced promoter. We used E. coli as the host organism, since it is widely used in recombinant processes. Our results show that the introduction of the self-regulatory circuit increases the soluble/insoluble ratio of recombinant protein at the expense of total protein yield. To further elucidate the dynamics of the system, we developed a computational model that is in agreement with the observed experimental data, and provides insight on the interplay between protein solubility and yield. Conclusion: Our work introduces the idea of a self-regulatory circuit for recombinant protein products, and paves the way for processes with reduced external control or monitoring needs. It demonstrates that the library of standard biological parts serves as a valuable resource for initial synthetic blocks that needs to be further refined to be successfully applied in practical problems of biotechnological significance. Finally, the development of a predictive model in conjunction with experimental validation facilitates a better understanding of the underlying dynamics and can be used as a guide to experimental design. http://www.jbioleng.org/content/6/1/2 Martin Dragosits Daniel Nicklas Ilias Tagkopoulos Journal of Biological Engineering 2012, null:2 2012-03-30T00:00:00Z doi:10.1186/1754-1611-6-2 /content/figures/1754-1611-6-2-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 2 2012-03-30T00:00:00Z PDF 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, null:4 2009-03-20T00:00:00Z doi:10.1186/1754-1611-3-4 /content/figures/1754-1611-3-4-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 4 2009-03-20T00:00:00Z XML 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, null:5 2008-04-14T00:00:00Z doi:10.1186/1754-1611-2-5 /content/figures/1754-1611-2-5-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 5 2008-04-14T00:00:00Z XML Tumor shape and size effect on drug delivery to solid tumors are studied, based on the application of thegoverning equations for fluid flow, i.e., the conservation laws for mass and momentum, to physiological systemscontaining solid tumors. The discretized form of the governing equations, with appropriate boundary conditions,is developed for predefined tumor geometries. The governing equations are solved using a numerical method, theelement-based finite volume method. Interstitial fluid pressure and velocity are used to show the details of drugdelivery in a solid tumor, under an assumption that drug particles flow with the interstitial fluid. Drug deliveryproblems have been most extensively researched in spherical tumors, which have been the simplest to examinewith the analytical methods. With our numerical method, however, more complex shapes of the tumor can bestudied. The numerical model of fluid flow in solid tumors previously introduced by our group is furtherdeveloped to incorporate and investigate non-spherical tumors such as prolate and oblate ones. Also the effects ofthe surface area per unit volume of the tissue, vascular and interstitial hydraulic conductivity on drug delivery areinvestigated. http://www.jbioleng.org/content/6/1/4 M Soltani P Chen Journal of Biological Engineering 2012, null:4 2012-04-25T00:00:00Z doi:10.1186/1754-1611-6-4 /content/figures/1754-1611-6-4-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 4 2012-04-25T00:00:00Z PDF 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, null:19 2009-10-29T00:00:00Z doi:10.1186/1754-1611-3-19 /content/figures/1754-1611-3-19-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 19 2009-10-29T00:00:00Z 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, null:9 2009-06-26T00:00:00Z doi:10.1186/1754-1611-3-9 /content/figures/1754-1611-3-9-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 9 2009-06-26T00:00:00Z XML Background: Biological Computer Aided Design (bioCAD) assists the de novo design and selection of existing genetic components to achieve a desired biological activity, as part of an integrated design-build-test cycle. To meet the emerging needs of Synthetic Biology, bioCAD tools must address the increasing prevalence of combinatorial library design, design rule specification, and scar-less multi-part DNA assembly. Results: We report the development and deployment of web-based bioCAD software, DeviceEditor, which provides a graphical design environment that mimics the intuitive visual whiteboard design process practiced in biological laboratories. The key innovations of DeviceEditor include visual combinatorial library design, direct integration with scar-less multi-part DNA assembly design automation, and a graphical user interface for the creation and modification of design specification rules. We demonstrate how biological designs are rendered on the DeviceEditor canvas, and we present effective visualizations of genetic component ordering and combinatorial variations within complex designs. Conclusions: DeviceEditor liberates researchers from DNA base-pair manipulation, and enables users to create successful prototypes using standardized, functional, and visual abstractions. Open and documented software interfaces support further integration of DeviceEditor with other bioCAD tools and software platforms. DeviceEditor saves researcher time and institutional resources through correct-by-construction design, the automation of tedious tasks, design reuse, and the minimization of DNA assembly costs. http://www.jbioleng.org/content/6/1/1 Joanna Chen Douglas Densmore Timothy Ham Jay Keasling Nathan Hillson Journal of Biological Engineering 2012, null:1 2012-02-28T00:00:00Z doi:10.1186/1754-1611-6-1 /content/figures/1754-1611-6-1-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 1 2012-02-28T00:00:00Z XML Bacterial cellulose has been used in the food industry for applications such as low-calorie desserts, salads, and fabricated foods. It has also been used in the paper manufacturing industry to enhance paper strength, the electronics industry in acoustic diaphragms for audio speakers, the pharmaceutical industry as filtration membranes, and in the medical field as wound dressing and artificial skin material. In this study, different types of plastic composite support (PCS) were implemented separately within a fermentation medium in order to enhance bacterial cellulose (BC) production by Acetobacter xylinum. The optimal composition of nutritious compounds in PCS was chosen based on the amount of BC produced. The selected PCS was implemented within a bioreactor to examine the effects on BC production in a batch fermentation. The produced BC was analyzed using X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), thermogravimetric analysis (TGA), and dynamic mechanical analysis (DMA). Among thirteen types of PCS, the type SFYR+ was selected as solid support for BC production by A. xylinum in a batch biofilm reactor due to its high nitrogen content, moderate nitrogen leaching rate, and sufficient biomass attached on PCS. The PCS biofilm reactor yielded BC production (7.05 g/L) that was 2.5-fold greater than the control (2.82 g/L). The XRD results indicated that the PCS-grown BC exhibited higher crystallinity (93%) and similar crystal size (5.2 nm) to the control. FESEM results showed the attachment of A. xylinum on PCS, producing an interweaving BC product. TGA results demonstrated that PCS-grown BC had about 95% water retention ability, which was lower than BC produced within suspended-cell reactor. PCS-grown BC also exhibited higher Tmax compared to the control. Finally, DMA results showed that BC from the PCS biofilm reactor increased its mechanical property values, i.e., stress at break and Young's modulus when compared to the control BC. The results clearly demonstrated that implementation of PCS within agitated fermentation enhanced BC production and improved its mechanical properties and thermal stability. http://www.jbioleng.org/content/3/1/12 Kuan-Chen Cheng Jeffrey Catchmark Ali Demirci Journal of Biological Engineering 2009, null:12 2009-07-24T00:00:00Z doi:10.1186/1754-1611-3-12 /content/figures/1754-1611-3-12-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 12 2009-07-24T00:00:00Z XML