BglBricks: A flexible standard for biological part assembly

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 <it>E. coli </it>genome.</p> <p>Conclusions</p> <p>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.</p

Seroprevalence and Risk Factors for Rickettsia and Leptospira Infection in Four Ecologically Distinct Regions of Peru.

Rickettsia and Leptospira spp. are under-recognized causes of acute febrile disease worldwide. Rickettsia species are often placed into the spotted fever group rickettsiae (SFGR) and typhus group rickettsiae (TGR). We explored the antibody prevalence among humans for these two groups of rickettsiae in four regions of Peru (Lima, Cusco, Puerto Maldonado, and Tumbes) and for Leptospira spp. in Puerto Maldonado and Tumbes. We also assessed risk factors for seropositivity and collected serum samples and ectoparasites from peri-domestic animals from households in sites with high human seroprevalence. In total, we tested 2,165 human sera for antibodies (IgG) against SFGR and TGR by ELISA and for antibodies against Leptospira by a microscopic agglutination test. Overall, human antibody prevalence across the four sites was 10.6% for SFGR (ranging from 6.2% to 14.0%, highest in Tumbes) and 3.3% for TGR (ranging from 2.6% to 6.4%, highest in Puerto Maldonado). Factors associated with seroreactivity against SFGR were male gender, older age, contact with backyard birds, and working in agriculture or with livestock. However, exposure to any kind of animal within the household decreased the odds ratio by half. Age was the only variable associated with higher TGR seroprevalence. The prevalence of Leptospira was 11.3% in Puerto Maldonado and 5.8% in Tumbes, with a borderline association with keeping animals in the household. We tested animal sera for Leptospira and conducted polymerase chain reaction (PCR) to detect Rickettsia species among ectoparasites collected from domestic animals in 63 households of seropositive participants and controls. We did not find any association between animal infection and human serostatus

BBF RFC 21: BglBricks Assembly Standard

The BglBricks standard has been developed as an alternative to the original XbaI/SpeI standard primarily to provide a solution to the issue of generating translational-fusion parts. When joined together in a standard assembly reaction, BglBricks parts contain a scar sequence of GGATCT between the two parts. This sequence conveniently translates as Gly-Ser in the zero frame, a commonly-used linker in protein engineering. BglBricks has previously been called the “BglBrick” standard, and is also referred to as Assembly standard 21. A consolidated description of the standard is available at: http://openwetware.org/wiki/Template:AndersonLab:BglBrick_Standar

Serologic Evidence of Scrub Typhus in the Peruvian Amazon.

Using a large, passive, febrile surveillance program in Iquitos, Peru, we retrospectively tested human blood specimens for scrub typhus group orientiae by ELISA, immunofluorescence assay, and PCR. Of 1,124 participants, 60 (5.3%) were seropositive, and 1 showed evidence of recent active infection. Our serologic data indicate that scrub typhus is present in the Peruvian Amazon

Eugene – A Domain Specific Language for Specifying and Constraining Synthetic Biological Parts, Devices, and Systems

BACKGROUND: Synthetic biological systems are currently created by an ad-hoc, iterative process of specification, design, and assembly. These systems would greatly benefit from a more formalized and rigorous specification of the desired system components as well as constraints on their composition. Therefore, the creation of robust and efficient design flows and tools is imperative. We present a human readable language (Eugene) that allows for the specification of synthetic biological designs based on biological parts, as well as provides a very expressive constraint system to drive the automatic creation of composite Parts (Devices) from a collection of individual Parts. RESULTS: We illustrate Eugene's capabilities in three different areas: Device specification, design space exploration, and assembly and simulation integration. These results highlight Eugene's ability to create combinatorial design spaces and prune these spaces for simulation or physical assembly. Eugene creates functional designs quickly and cost-effectively. CONCLUSIONS: Eugene is intended for forward engineering of DNA-based devices, and through its data types and execution semantics, reflects the desired abstraction hierarchy in synthetic biology. Eugene provides a powerful constraint system which can be used to drive the creation of new devices at runtime. It accomplishes all of this while being part of a larger tool chain which includes support for design, simulation, and physical device assembly

Rickettsial Disease in the Peruvian Amazon Basin.

Using a large, passive, clinic-based surveillance program in Iquitos, Peru, we characterized the prevalence of rickettsial infections among undifferentiated febrile cases and obtained evidence of pathogen transmission in potential domestic reservoir contacts and their ectoparasites. Blood specimens from humans and animals were assayed for spotted fever group rickettsiae (SFGR) and typhus group rickettsiae (TGR) by ELISA and/or PCR; ectoparasites were screened by PCR. Logistic regression was used to determine associations between patient history, demographic characteristics of participants and symptoms, clinical findings and outcome of rickettsial infection. Of the 2,054 enrolled participants, almost 2% showed evidence of seroconversion or a 4-fold rise in antibody titers specific for rickettsiae between acute and convalescent blood samples. Of 190 fleas (Ctenocephalides felis) and 60 ticks (Rhipicephalus sanguineus) tested, 185 (97.4%) and 3 (5%), respectively, were positive for Rickettsia spp. Candidatus Rickettsia asemboensis was identified in 100% and 33% of the fleas and ticks tested, respectively. Collectively, our serologic data indicates that human pathogenic SFGR are present in the Peruvian Amazon and pose a significant risk of infection to individuals exposed to wild, domestic and peri-domestic animals and their ectoparasites

Targeted full-genome amplification and sequencing of dengue virus types 1–4 from South America

International audienceWe report optimized workflows for full-genome sequencing of dengue viruses (DENVs) 1–4. Based on alignments of publicly available complete genomes we modified and expanded existing primers sets to amplify DENV genotypes that were previously difficult or impossible to sequence. We also report improvements to streamline laboratory handling, including a dual amplification strategy for easy and difficult to sequence “high-copy” and “low-copy” templates, respectively, and normalization of PCR cycling conditions across serotypes. High-copy templates can be sequenced following amplification of as few as 5 overlapping segments covering the complete viral genome, whereas low-copy templates can be sequenced following amplification of no more than 10 overlapping segments of smaller size. These changes have been validated using a balanced set of wild-type DENV genomes (11 of DENV1, 14 of DENV2, 13 of DENV3 and 7 of DENV4) derived from human serum samples collected throughout South America over the past 15 years. The changes described enable generation of complete DENV genomes from wild-type samples without the need for viral enrichment via passaging through laboratory cell lines. This should facilitate quick and cost-effective generation of DENV full-genome sequences of the type needed for accurate epidemiological surveillance and thorough evolutionary studies of wild-type DENVs