http://www.jbioleng.org The latest research articles published by Journal of Biological Engineering 2011-12-16T00:00:00Z A modified BioBrickTM assembly method was developed with higher fidelity than current protocols. The method utilizes a PCR reaction with a standard primer set to amplify the inserted part. Background colonies are reduced by a combination of dephosphorylation and digestion with DpnI restriction endonuclease to reduce vector and insert background respectively. The molar ratio of the insert to vector in the ligation was also optimized, with the accuracy of the transformed construct approaching 100%. http://www.jbioleng.org/content/5/1/17 Michael Speer Tom Richard Journal of Biological Engineering 2011, null:17 2011-12-16T00:00:00Z doi:10.1186/1754-1611-5-17 /content/figures/1754-1611-5-17-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 17 2011-12-16T00:00:00Z PDF Background: In a globalized word, prevention of infectious diseases is a major challenge. Rapid detection of viable virus particles in water and other environmental samples is essential to public health risk assessment, homeland security and environmental protection. Current virus detection methods, especially assessing viral infectivity, are complex and time-consuming, making point-of-care detection a challenge. Faster, more sensitive, highly specific methods are needed to quantify potentially hazardous viral pathogens and to determine if suspected materials contain viable viral particles. Fourier transform infrared (FTIR) spectroscopy combined with cellular-based sensing, may offer a precise way to detect specific viruses. This approach utilizes infrared light to monitor changes in molecular components of cells by tracking changes in absorbance patterns produced following virus infection. In this work poliovirus (PV1) was used to evaluate the utility of FTIR spectroscopy with cell culture for rapid detection of infective virus particles. Results: Buffalo green monkey kidney (BGMK) cells infected with different virus titers were studied at 1 - 12 hours post-infection (h.p.i.). A partial least squares (PLS) regression method was used to analyze and model cellular responses to different infection titers and times post-infection. The model performs best at 8 h.p.i., resulting in an estimated root mean square error of cross validation (RMSECV) of 17 plaque forming units (PFU)/ml when using low titers of infection of 10 and 100 PFU/ml. Higher titers, from 103 to 106 PFU/ml, could also be reliably detected. Conclusions: This approach to poliovirus detection and quantification using FTIR spectroscopy and cell culture could potentially be extended to compare biochemical cell responses to infection with different viruses. This virus detection method could feasibly be adapted to an automated scheme for use in areas such as water safety monitoring and medical diagnostics. http://www.jbioleng.org/content/5/1/16 Felipe Lee-Montiel Kelly Reynolds Mark Riley Journal of Biological Engineering 2011, null:16 2011-12-05T00:00:00Z doi:10.1186/1754-1611-5-16 /content/figures/1754-1611-5-16-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 16 2011-12-05T00:00:00Z XML CD-ELISA uses the microfluidic ranking method and centrifugal force to control the testing solution as it flows into the reaction region. The most challenging part of CD-ELISA is controlling the flow process for different biological testing solutions, i.e. the controlling sequence for the microfluidic channel valves. The microfluidic channel valve is therefore the most important fluid channel structure for CD-ELISA. In this study, we propose a valve design suitable for a wide range rotational speeds which can be applied for mass production (molding). Together with supporting experiments, simulation based on two-phase flow theory is used in this study, and the feasibility of this novel valve design is confirmed. Influencing design factors for the microfluidic channel valves in CD-ELISA are investigated, including various shapes of the arc, distance d, radius r, the location of the center of the circle, and the contact angle. From both the experimental results and the simulated results, it is evident that the narrowest channel width and the contact angle are the primary factors influencing valve burst frequency. These can be used as the main controlling factors during the design. http://www.jbioleng.org/content/5/1/15 Samuel I En Lin Journal of Biological Engineering 2011, null:15 2011-11-07T00:00:00Z doi:10.1186/1754-1611-5-15 /content/figures/1754-1611-5-15-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 15 2011-11-07T00:00:00Z XML On February 15, 2008, the National Academy of Engineering unveiled their list of 14 Grand Challenges for Engineering. Building off of tremendous advancements in the past century, these challenges were selected for their role in assuring a sustainable existence for the rapidly increasing global community. It is no accident that the first five Challenges on the list involve the development of sustainable energy sources and management of environmental resources. While the focus of this review is to address the single Grand Challenge of "develop carbon sequestration methods", is will soon be clear that several other Challenges are intrinsically tied to it through the principles of sustainability. How does the realm of biological engineering play a role in addressing these Grand Challenges? http://www.jbioleng.org/content/5/1/14 Ben Stuart Journal of Biological Engineering 2011, null:14 2011-11-02T00:00:00Z doi:10.1186/1754-1611-5-14 /content/figures/1754-1611-5-14-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 14 2011-11-02T00:00:00Z XML Background: The threatened plant Centella asiatica L. is traditionallyused for a number of remedies. In vitro plant propagation and enhanced metabolite production of active metabolites through biotechnological approaches has gained attention in recent years. Results: Present study reveals that 6-benzyladenine (BA) either alone or in combination with 1-naphthalene acetic acid (NAA) supplemented in Murashige and Skoog (MS) medium at different concentrations produced good quality callus from leaf explants of C. asiatica. The calli produced on different plant growth regulators at different concentrations were mostly embryogenic and green. Highest shoot regeneration efficiency; 10 shoots per callus explant, from non-embryogenic callus was observed on 4.42 μM BA with 5.37 μM NAA. Best rooting response was observed at 5.37 and 10.74 μM NAA with 20 average number of roots per explant. Calli and regenerated plants extracts inhibited bacterial growth with mean zone of inhibition 9-13 mm diameter when tested against six bacterial strains using agar well diffusion method. Agar tube dilution method for antifungal assay showed 3.2-76% growth inhibition of Mucor species, Aspergillus fumigatus and Fusarium moliniformes. Conclusions: The present investigation reveals that non-embryogenic callus can be turned into embryos and plantlets if cultured on appropriate medium. Furthermore, callus from leaf explant of C. asiatica can be a good source for production of antimicrobial compounds through bioreactor. http://www.jbioleng.org/content/5/1/13 Yamin Bibi Muhammad Zia Sobia Nisa Darima Habib Abdul Waheed Fayyaz Chaudhary Journal of Biological Engineering 2011, null:13 2011-10-12T00:00:00Z doi:10.1186/1754-1611-5-13 /content/figures/1754-1611-5-13-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 13 2011-10-12T00:00:00Z XML Background: As engineered biological systems become more complex, it is increasingly common to express multiple operons from different plasmids and inducible expression systems within a single host cell. Optimizing such systems often requires screening combinations of origins of replication, expression systems, and antibiotic markers. This procedure is hampered by a lack of quantitative data on how these components behave when more than one origin of replication or expression system are used simultaneously. Additionally, this process can be time consuming as it often requires the creation of new vectors or cloning into existing but disparate vectors. Results: Here, we report the development and characterization of a library of expression vectors compatible with the BglBrick standard (BBF RFC 21). We have designed and constructed 96 BglBrick-compatible plasmids with a combination of replication origins, antibiotic resistance genes, and inducible promoters. These plasmids were characterized over a range of inducer concentrations, in the presence of non-cognate inducer molecules, and with several growth media, and their characteristics were documented in a standard format datasheet. A three plasmid system was used to investigate the impact of multiple origins of replication on plasmid copy number. Conclusions: The standardized collection of vectors presented here allows the user to rapidly construct and test the expression of genes with various combinations of promoter strength, inducible expression system, copy number, and antibiotic resistance. The quantitative datasheets created for these vectors will increase the predictability of gene expression, especially when multiple plasmids and inducers are utilized. http://www.jbioleng.org/content/5/1/12 Taek Soon Lee Rachel Krupa Fuzhong Zhang Meghdad Hajimorad William Holtz Nilu Prasad Sung Kuk Lee Jay Keasling Journal of Biological Engineering 2011, null:12 2011-09-20T00:00:00Z doi:10.1186/1754-1611-5-12 /content/figures/1754-1611-5-12-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 12 2011-09-20T00:00:00Z XML Background: Stink bugs (Hemiptera: Pentatomidae) comprise a critically important insect pest complex affecting 12 major crops worldwide including cotton. In the US, stink bug damage to developing cotton bolls causes boll abscission, lint staining, reduced fiber quality, and reduced yields with estimated losses ranging from 10 to 60 million dollars annually. Unfortunately, scouting for stink bug damage in the field is laborious and excessively time consuming. To improve scouting accuracy and efficiency, we investigated fluorescence changes in cotton boll tissues as a result of stink bug feeding. Results: Fluorescent imaging under long-wave ultraviolet light showed that stink bug-damaged lint, the inner carpal wall, and the outside of the boll emitted strong blue-green fluorescence in a circular region near the puncture wound, whereas undamaged tissue emissions occurred at different wavelengths; the much weaker emission of undamaged tissue was dominated by chlorophyll fluorescence. We further characterized the optimum emission and excitation spectra to distinguish between stink bug damaged bolls from undamaged bolls. Conclusions: The observed characteristic fluorescence peaks associated with stink bug damage give rise to a fluorescence-based method to rapidly distinguish between undamaged and stink bug damaged cotton bolls. Based on the fluorescent fingerprint, we envision a fluorescence reflectance imaging or a fluorescence ratiometric device to assist pest management professionals with rapidly determining the extent of stink bug damage in a cotton field. http://www.jbioleng.org/content/5/1/11 Jinjun Xia Adnan Mustafic Michael Toews Mark Haidekker Journal of Biological Engineering 2011, null:11 2011-08-04T00:00:00Z doi:10.1186/1754-1611-5-11 /content/figures/1754-1611-5-11-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 11 2011-08-04T00:00:00Z XML Background: Engineers frequently vary design parameters to optimize the behaviour of a system. However, synthetic biologists lack the tools to rapidly explore a critical design parameter, gene expression level, and have no means of systematically varying the dosage of an entire genetic circuit. As a step toward overcoming this shortfall, we have developed a technology that enables the same plasmid to be maintained at different copy numbers in a set of closely related cells. This provides a rapid method for exploring gene or cassette dosage effects. Results: We engineered two sets of strains to constitutively provide a trans-acting replication factor, either Pi of the R6K plasmid or RepA of the ColE2 plasmid, at different doses. Each DIAL (different allele) strain supports the replication of a corresponding plasmid at a constant level between 1 and 250 copies per cell. The plasmids exhibit cell-to-cell variability comparable to other popular replicons, but with improved stability. Since the origins are orthogonal, both replication factors can be incorporated into the same cell. We demonstrate the utility of these strains by rapidly assessing the optimal expression level of a model biosynthetic pathway for violecein. Conclusions: The DIAL strains can rapidly optimize single gene expression levels, help balance expression of functionally coupled genetic elements, improve investigation of gene and circuit dosage effects, and enable faster development of metabolic pathways. http://www.jbioleng.org/content/5/1/10 Joshua Kittleson Sherine Cheung J Christopher Anderson Journal of Biological Engineering 2011, null:10 2011-07-25T00:00:00Z doi:10.1186/1754-1611-5-10 /content/figures/1754-1611-5-10-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 10 2011-07-25T00:00:00Z XML Members of the synthetic biology community have discussed the significance of word selection when describing synthetic biology to the general public. In particular, many leaders proposed the word "create" was laden with negative connotations. We found that word choice and framing does affect public perception of synthetic biology. In a controlled experiment, participants perceived synthetic biology more negatively when "create" was used to describe the field compared to "construct" (p = 0.008). Contrary to popular opinion among synthetic biologists, however, low religiosity individuals were more influenced negatively by the framing manipulation than high religiosity people. Our results suggest that synthetic biologists directly influence public perception of their field through avoidance of the word "create". http://www.jbioleng.org/content/5/1/9 Brianna Pearson Sam Snell Kyri Bye-Nagel Scott Tonidandel Laurie Heyer A. Malcolm Campbell Journal of Biological Engineering 2011, null:9 2011-07-21T00:00:00Z doi:10.1186/1754-1611-5-9 /content/figures/1754-1611-5-9-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 9 2011-07-21T00:00:00Z XML Background: Bacterial cell lysis is a widely studied mechanism that can be achieved through the intracellular expression of phage native lytic proteins. This mechanism can be exploited for programmed cell death and for gentle cell disruption to release recombinant proteins when in vivo secretion is not feasible. Several genetic parts for cell lysis have been developed and their quantitative characterization is an essential step to enable the engineering of synthetic lytic systems with predictable behavior. Results: Here, a BioBrick™ lysis device present in the Registry of Standard Biological Parts has been quantitatively characterized. Its activity has been measured in E. coli by assembling the device under the control of a well characterized N-3-oxohexanoyl-L-homoserine lactone (HSL) -inducible promoter and the transfer function, lysis dynamics, protein release capability and genotypic and phenotypic stability of the device have been evaluated. Finally, its modularity was tested by assembling the device to a different inducible promoter, which can be triggered by heat induction. Conclusions: The studied device is suitable for recombinant protein release as 96% of the total amount of the intracellular proteins was successfully released into the medium. Furthermore, it has been shown that the device can be assembled to different input devices to trigger cell lysis in response to a user-defined signal. For this reason, this lysis device can be a useful tool for the rational design and construction of complex synthetic biological systems composed by biological parts with known and well characterized function. Conversely, the onset of mutants makes this device unsuitable for the programmed cell death of a bacterial population. http://www.jbioleng.org/content/5/1/8 Lorenzo Pasotti Susanna Zucca Manuel Lupotto Maria Gabriella Cusella De Angelis Paolo Magni Journal of Biological Engineering 2011, null:8 2011-06-07T00:00:00Z doi:10.1186/1754-1611-5-8 /content/figures/1754-1611-5-8-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 8 2011-06-07T00:00:00Z XML