http://www.jbioleng.org The latest research articles published by Journal of Biological Engineering 2013-05-10T00:00:00Z Background: Realizing constructive applications of synthetic biology requires continued development of enabling technologies as well as policies and practices to ensure these technologies remain accessible for research. Broadly defined, enabling technologies for synthetic biology include any reagent or method that, alone or in combination with associated technologies, provides the means to generate any new research tool or application. Because applications of synthetic biology likely will embody multiple patented inventions, it will be important to create structures for managing intellectual property rights that best promote continued innovation. Monitoring the enabling technologies of synthetic biology will facilitate the systematic investigation of property rights coupled to these technologies and help shape policies and practices that impact the use, regulation, patenting, and licensing of these technologies. Results: We conducted a survey among a self-identifying community of practitioners engaged in synthetic biology research to obtain their opinions and experiences with technologies that support the engineering of biological systems. Technologies widely used and considered enabling by survey participants included public and private registries of biological parts, standard methods for physical assembly of DNA constructs, genomic databases, software tools for search, alignment, analysis, and editing of DNA sequences, and commercial services for DNA synthesis and sequencing. Standards and methods supporting measurement, functional composition, and data exchange were less widely used though still considered enabling by a subset of survey participants. Conclusions: The set of enabling technologies compiled from this survey provide insight into the many and varied technologies that support innovation in synthetic biology. Many of these technologies are widely accessible for use, either by virtue of being in the public domain or through legal tools such as non-exclusive licensing. Access to some patent protected technologies is less clear and use of these technologies may be subject to restrictions imposed by material transfer agreements or other contract terms. We expect the technologies considered enabling for synthetic biology to change as the field advances. By monitoring the enabling technologies of synthetic biology and addressing the policies and practices that impact their development and use, our hope is that the field will be better able to realize its full potential. http://www.jbioleng.org/content/7/1/13 Linda Kahl Drew Endy Journal of Biological Engineering 2013, null:13 2013-05-10T00:00:00Z doi:10.1186/1754-1611-7-13 /content/figures/1754-1611-7-13-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 13 2013-05-10T00:00:00Z PDF Background: The chromosomal integration of biological parts in the host genome enables the engineering of plasmid-free stable strains with single-copy insertions of the desired gene networks. Although different integrative vectors were proposed, no standard pre-assembled genetic tool is available to carry out this task. Synthetic biology concepts can contribute to the development of standardized and user friendly solutions to easily produce engineered strains and to rapidly characterize the desired genetic parts in single-copy context. Results: In this work we report the design of a novel integrative vector that allows the genomic integration of biological parts compatible with the RFC10, RFC23 and RFC12 BioBrick™ standards in Escherichia coli. It can also be specialized by using BioBrick™ parts to target the desired integration site in the host genome. The usefulness of this vector has been demonstrated by integrating a set of BioBrick™ devices in two different loci of the E. coli chromosome and by characterizing their activity in single-copy. Construct stability has also been evaluated and compared with plasmid-borne solutions. Conclusions: Physical modularity of biological parts has been successfully applied to construct a ready-to-engineer BioBrick™ vector, suitable for a stable chromosomal insertion of standard parts via the desired recombination method, i.e. the bacteriophage integration mechanism or homologous recombination. In contrast with previously proposed solutions, it is a pre-assembled vector containing properly-placed restriction sites for the direct transfer of various formats of BioBrick™ parts. This vector can facilitate the characterization of parts avoiding copy number artefacts and the construction of antibiotic resistance-free engineered microbes, suitable for industrial use. http://www.jbioleng.org/content/7/1/12 Susanna Zucca Lorenzo Pasotti Nicolò Politi Maria Cusella De Angelis Paolo Magni Journal of Biological Engineering 2013, null:12 2013-05-10T00:00:00Z doi:10.1186/1754-1611-7-12 /content/figures/1754-1611-7-12-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 12 2013-05-10T00:00:00Z XML Endothelialization of therapeutic cardiovascular implants is essential for their intravascular hemocompatibility. We previously described a novel nanowell-RGD-nanoparticle ensemble, which when applied to surfaces led to enhanced endothelialization and retention under static conditions and low flow rates. In the present study we extend our work to determine the interrelated effects of flow rate and the orientation of ensemble-decorated surface arrays on the growth, adhesion and morphology of endothelial cells. Human umbilical vascular endothelial cells (HUVECs) were grown on array surfaces with either 1 μm × 5 μm spacing (“parallel to flow”) and 5 μm × 1 μm spacing (“perpendicular to flow”) and were exposed to a range of shear stress of (0 to 4.7 ± 0.2 dyn·cm-2 ), utilizing a pulsatile flow chamber. Under physiological flow (4.7 ± 0.2 dyn·cm-2), RGD-nanoparticle-nanowell array patterning significantly enhanced cell adhesion and spreading compared with control surfaces and with static conditions. Furthermore, improved adhesion coincided with higher alignment to surface patterning, intimating the importance of interaction and response to the array surface as a means of resisting flow detachment. Under sub-physiological condition (1.7 ± 0.3 dyn·cm-2; corresponding to early angiogenesis), nanowell-nanoparticle patterning did not provide enhanced cell growth and adhesion compared with control surfaces. However, it revealed increased alignment along the direction of flow, rather than the direction of the pattern, thus potentially indicating a threshold for cell guidance and related retention. These results could provide a cue for controlling cell growth and alignment under varying physiological conditions. http://www.jbioleng.org/content/7/1/11 Katherine McCracken Phat Tran David You Marvin Slepian Jeong-Yeol Yoon Journal of Biological Engineering 2013, null:11 2013-04-22T00:00:00Z doi:10.1186/1754-1611-7-11 /content/figures/1754-1611-7-11-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 11 2013-04-22T00:00:00Z XML Background: Cyanobacteria, prokaryotic cells with oxygenic photosynthesis, are excellent bioengineering targets to convert solar energy into solar fuels. Tremendous genetic engineering approaches and tools have been and still are being developed for prokaryotes. However, the progress for cyanobacteria is far behind with a specific lack of non-native inducible promoters. Results: We report the development of engineered TetR-regulated promoters with a wide dynamic range of transcriptional regulation. An optimal 239 (±16) fold induction in darkness (white-light-activated heterotrophic growth, 24 h) and an optimal 290 (±93) fold induction in red light (photoautotrophic growth, 48 h) were observed with the L03 promoter in cells of the unicellular cyanobacterium Synechocystis sp. strain ATCC27184 (i.e. glucose-tolerant Synechocystis sp. strain PCC 6803). By altering only few bases of the promoter in the narrow region between the -10 element and transcription start site significant changes in the promoter strengths, and consequently in the range of regulations, were observed. Conclusions: The non-native inducible promoters developed in the present study are ready to be used to further explore the notion of custom designed cyanobacterial cells in the complementary frameworks of metabolic engineering and synthetic biology. http://www.jbioleng.org/content/7/1/10 Hsin-Ho Huang Peter Lindblad Journal of Biological Engineering 2013, null:10 2013-04-22T00:00:00Z doi:10.1186/1754-1611-7-10 /content/figures/1754-1611-7-10-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 10 2013-04-22T00:00:00Z XML Background: Embryonic stem cells (ESCs) have been implicated to have tremendous impact in regenerative therapeutics of various diseases, including Type 1 Diabetes. Upon generation of functionally mature ESC derived islet-like cells, they need to be implanted into diabetic patients to restore the loss of islet activity. Encapsulation in alginate microcapsules is a promising route of implantation, which can protect the cells from the recipient’s immune system. While there has been a significant investigation into islet encapsulation over the past decade, the feasibility of encapsulation and differentiation of ESCs has been less explored. Research over the past few years has identified the cellular mechanical microenvironment to play a central role in phenotype commitment of stem cells. Therefore it will be important to design the encapsulation material to be supportive to cellular functionality and maturation. Results: This work investigated the effect of stiffness of alginate substrate on initial differentiation and phenotype commitment of murine ESCs. ESCs grown on alginate substrates tuned to similar biomechanical properties of native pancreatic tissue elicited both an enhanced and incrementally responsive differentiation towards endodermal lineage traits. Conclusions: The insight into these biophysical phenomena found in this study can be used along with other cues to enhance the differentiation of embryonic stem cells toward a specific lineage fate. http://www.jbioleng.org/content/7/1/9 Joseph Candiello Satish Singh Keith Task Prashant Kumta Ipsita Banerjee Journal of Biological Engineering 2013, null:9 2013-04-09T00:00:00Z doi:10.1186/1754-1611-7-9 /content/figures/1754-1611-7-9-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 9 2013-04-09T00:00:00Z XML Background: Bacterial contamination on touch surfaces results in increased risk of infection. In the last few decades, work has been done on the antimicrobial properties of copper and its alloys against a range of micro-organisms threatening public health in food processing, healthcare and air conditioning applications; however, an optimum copper method of surface deposition and mass structure has not been identified. Results: A proof-of-concept study of the disinfection effectiveness of three copper surfaces was performed. The surfaces were produced by the deposition of copper using three methods of thermal spray, namely, plasma spray, wire arc spray and cold spray The surfaces were then inoculated with meticillin-resistant Staphylococcus aureus (MRSA). After a two hour exposure to the surfaces, the surviving MRSA were assayed and the results compared.The differences in the copper depositions produced by the three thermal spray methods were examined in order to explain the mechanism that causes the observed differences in MRSA killing efficiencies. The cold spray deposition method was significantly more effective than the other methods. It was determined that work hardening caused by the high velocity particle impacts created by the cold spray technique results in a copper microstructure that enhances ionic diffusion, and copper ions are principally responsible for antimicrobial activity. Conclusions: This test showed significant microbiologic differences between coatings produced by different spray techniques and demonstrates the importance of the copper application technique. The cold spray technique shows superior anti-microbial effectiveness caused by the high impact velocity imparted to the sprayed particles which results in high dislocation density and high ionic diffusivity. http://www.jbioleng.org/content/7/1/8 Victor Champagne Dennis Helfritch Journal of Biological Engineering 2013, null:8 2013-03-27T00:00:00Z doi:10.1186/1754-1611-7-8 /content/figures/1754-1611-7-8-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 8 2013-03-27T00:00:00Z XML Background: The pluripotency and self renewing properties of human embryonic stem cells (hESC) make them a valuable tool in the fields of developmental biology, pharmacology and regenerative medicine. Therefore, there exists immense interest in devising strategies for hESC propagation and differentiation. Methods involving simulation of the native stem cell microenvironment, both chemical and physical, have received a lot of attention in recent years. Equally important is evidence that cells can also sense the mechanical properties of their microenvironment. In this study, we test the hypothesis that hESCs accept mechanical cues for differentiation from the substrate by culturing them on flexible polydimethylsiloxane (PDMS) of varying stiffness. Results: PDMS substrates were prepared using available commercial formulations and characterized for stiffness, surface properties and efficiency of cell attachment and proliferation. Across different substrate stiffness, cell numbers, cell attachment and cell surface area were found to be similar. Expression of pluripotency markers decreased with increased time in culture across all PDMS substrates of varying stiffness. Analysis of gene expression of differentiation markers indicates that the differentiation process becomes less stochastic with longer culture times. Conclusions: We evaluated the utility of PDMS substrates for stem cell propagation and substrate mediated differentiation. The stiffness affected gene expression of pluripotent and differentiation markers with results indicating that these substrate systems could potentially be used to direct hESC fate towards early mesodermal lineages. This study suggests that coupled with soluble factors, PDMS substrates could potentially be useful in generating defined populations of differentiated cells. http://www.jbioleng.org/content/7/1/7 Nikolai Eroshenko Rukmani Ramachandran Vamsi Yadavalli Raj Rao Journal of Biological Engineering 2013, null:7 2013-03-21T00:00:00Z doi:10.1186/1754-1611-7-7 /content/figures/1754-1611-7-7-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 7 2013-03-21T00:00:00Z XML Among the important principles in biology that should be taught in biological engineering educational programs is the principle of optimization, what it means, why it is important, and how it comes about. This material can be presented at numerous levels throughout the curriculum. Understanding of this principle can lead biological engineers to expect it in many, if not all, biological system applications. Understanding optimization in biological systems can help understand the predictive power of evolutionary principles and what to expect from living things incorporated in designs. http://www.jbioleng.org/content/7/1/6 Arthur Johnson Journal of Biological Engineering 2013, null:6 2013-02-20T00:00:00Z doi:10.1186/1754-1611-7-6 /content/figures/1754-1611-7-6-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 6 2013-02-20T00:00:00Z XML Background: Stink bugs represent a major agricultural pest complex attacking more than 200 wild and cultivated plants,including cotton in the southeastern US. Stink bug feeding on developing cotton bolls will cause boll abortionor lint staining and thus reduced yield and lint value. Current methods for stink bug detection involve manualharvesting and cracking open of a sizable number of immature cotton bolls for visual inspection. This processis cumbersome, time consuming, and requires a moderate level of experience to obtain accurate estimates. Toimprove detection of stink bug feeding, we present here a method based on fluorescent imaging andsubsequent image analyses to determine the likelihood of stink bug damage in cotton bolls. Results: Damage to different structures of cotton bolls including lint and carpal wall can be observed under blueLED-induced fluorescence. Generally speaking, damaged regions fluoresce green, whereas non-damagedregions with chlorophyll fluoresce red. However, similar fluorescence emission is also observable on cottonbolls that have not been fed upon by stink bugs. Criteria based on fluorescent intensity and the size of thefluorescent spot allow to differentiate between true positives (fluorescent regions associated with stink bugfeeding) and false positives (fluorescent regions due to other causes). We found a detection rates with twocombined criteria of 87% for true-positive marks and of 8% for false-positive marks. Conclusions: The imaging technique presented herein gives rise to a possible detection apparatus where a cotton boll isimaged in the field and images processed by software. The unique fluorescent signature left by stink bugs canbe used to determine with high probability if a cotton boll has been punctured by a stink bug. We believe thistechnique, when integrated in a suitable device, could be used for more accurate detection in the field andallow for more optimized application of pest control. http://www.jbioleng.org/content/7/1/5 Adnan Mustafic Erin Roberts Michael Toews Mark Haidekker Journal of Biological Engineering 2013, null:5 2013-02-20T00:00:00Z doi:10.1186/1754-1611-7-5 /content/figures/1754-1611-7-5-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 5 2013-02-20T00:00:00Z PDF Background: Like other bacteria, Escherichia coli must carefully regulate the intracellular concentration of sodium ion (Na+). During the bacterial production of any organic acid, cations like Na+ invariably accumulate during a process which must maintain a near neutral pH. In this study, the E. coli nhaA gene encoding the Na+/H+ antiporter membrane protein and the nhaR gene encoding the NhaA regulatory protein were overexpressed in wild-type E. coli MG1655 and in MG1655 pflB (ALS1317) which lacks pyruvate formate lyase activity and thus accumulates lactate under anaerobic conditions. Results: Expression of either the nhaA or nhaR gene on the high copy inducible expression vector pTrc99A caused a significant reduction in the growth rate of MG1655. No change in growth rate was observed for MG1655 or ALS1317 for Na+ concentrations of 0.75–0.90 M when the medium copy pBR322 plasmid was used to overexpress the two genes. In a fed-batch process to produce the model acid lactate with NaOH addition for pH control, lactate accumulation ceased in MG1655, MG1655/pBR322, MG1655/pBR322-nhaR and MG1655/pBR322-nhaA when the concentration reached 55–58 g/L. In an identical process lactate accumulation in MG1655/pBR322-nhaAR did not terminate until the concentration reached over 70 g/L. Conclusions: Although overexpression the genes did not improve growth rate at high Na+ concentrations, the overexpression of nhaA and nhaR together led to a 25% increase in lactate production. Thus, the observed (absence of) impact that these genetic modifications had on growth rate is a poor indicator of their effect on acid accumulation. The overexpression of nhaAR did not cause faster lactate production, but permitted the culture to continue accumulating lactate at 10% greater Na+ concentration. http://www.jbioleng.org/content/7/1/3 Xianghao Wu Ronni Altman Mark Eiteman Elliot Altman Journal of Biological Engineering 2013, null:3 2013-01-29T00:00:00Z doi:10.1186/1754-1611-7-3 /content/figures/1754-1611-7-3-toc.gif Journal of Biological Engineering 1754-1611 ${item.volume} 3 2013-01-29T00:00:00Z XML