Automated robotic liquid handling assembly of modular DNA devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available "design space" represented by "devices" created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction. Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols. The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation

ALDH1A2 (RALDH2) genetic variation in human congenital heart disease

Abstract\ud \ud \ud \ud Background\ud \ud Signaling by the vitamin A-derived morphogen retinoic acid (RA) is required at multiple steps of cardiac development. Since conversion of retinaldehyde to RA by retinaldehyde dehydrogenase type II (ALDH1A2, a.k.a RALDH2) is critical for cardiac development, we screened patients with congenital heart disease (CHDs) for genetic variation at the ALDH1A2 locus.\ud \ud \ud \ud Methods\ud \ud One-hundred and thirty-three CHD patients were screened for genetic variation at the ALDH1A2 locus through bi-directional sequencing. In addition, six SNPs (rs2704188, rs1441815, rs3784259, rs1530293, rs1899430) at the same locus were studied using a TDT-based association approach in 101 CHD trios. Observed mutations were modeled through molecular mechanics (MM) simulations using the AMBER 9 package, Sander and Pmemd programs. Sequence conservation of observed mutations was evaluated through phylogenetic tree construction from ungapped alignments containing ALDH8 s, ALDH1Ls, ALDH1 s and ALDH2 s. Trees were generated by the Neighbor Joining method. Variations potentially affecting splicing mechanisms were cloned and functional assays were designed to test splicing alterations using the pSPL3 splicing assay.\ud \ud \ud \ud Results\ud \ud We describe in Tetralogy of Fallot (TOF) the mutations Ala151Ser and Ile157Thr that change non-polar to polar residues at exon 4. Exon 4 encodes part of the highly-conserved tetramerization domain, a structural motif required for ALDH oligomerization. Molecular mechanics simulation studies of the two mutations indicate that they hinder tetramerization. We determined that the SNP rs16939660, previously associated with spina bifida and observed in patients with TOF, does not affect splicing. Moreover, association studies performed with classical models and with the transmission disequilibrium test (TDT) design using single marker genotype, or haplotype information do not show differences between cases and controls.\ud \ud \ud \ud Conclusion\ud \ud In summary, our screen indicates that ALDH1A2 genetic variation is present in TOF patients, suggesting a possible causal role for this gene in rare cases of human CHD, but does not support the hypothesis that variation at the ALDH1A2 locus is a significant modifier of the risk for CHD in humans.Work supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 01/000090; 00/030722; 01/142381; 02/113402; 03/099982; 04/116068; 04/157044 and Conselho Nacional de Desenvolvimento Científico e Tecnológico 481872/20078. We would like to thank the careful work and thoughtful suggestions of the two reviewers responsible for the reviewing editorial process.Work supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 01/00009-0; 00/03072-2; 01/14238-1; 02/11340-2; 03/09998-2; 04/11606-8; 04/15704-4 and Conselho Nacional de Desenvolvimento Científico e Tecnológico 481872/2007-8. We would like to thank the careful work and thoughtful suggestions of the two reviewers responsible for the reviewing editorial process

Building a global alliance of biofoundries (vol 10, 2040, 2019)

The original version of this Comment contained errors in the legend of Figure 2, in which the locations of the fifteenth and sixteenth GBA members were incorrectly given as '(15) Australian Genome Foundry, Macquarie University; (16) Australian Foundry for Advanced Biomanufacturing, University of Queensland.'. The correct version replaces this with '(15) Australian Foundry for Advanced Biomanufacturing (AusFAB), University of Queensland and (16) Australian Genome Foundry, Macquarie University'. This has been corrected in both the PDF and HTML versions of the Comment

RALDH2 genetic variants and congenital heart disease.

Nós investigamos o papel da variação genética do gene RALDH2 e as doenças cardíacas congênitas (DCCs). Seis SNPs foram utilizados em um estudo de TDT. Testes de associação foram desenvolvidos e tanto os marcadores testados quanto os haplótipos analisados não mostraram associação com a doença. Análise do polimorfismo A151G indica que a variante produz mudanças substanciais na estrutura do RNAm. Esta variante está localizada em um exonic splicing enhancer (ESE). Estudos funcionais de splicing não mostraram impacto significante desta variante sobre a alteração do splicing do gene. Este estudo foi aplicado à outra mutação (G151T) encontrada no exon 4 durante o sequenciamento do gene RALDH2 e mostrou aumento no sinal de splicing. Nós encontramos mais quatro mutações: rs34645259 (5\'UTR), T157G (exon 4), rs4646626 (exon 9) e rs35251510 (exon 11). Em resumo, não foi encontrada associação entre DCCs e variações genéticas no gene RALDH2. As mutações encontradas deverão ser analisadas funcionalmente de forma a definir seu papel na perturbação da via do AR em humanos.The aim of the study was to investigate the role of genetic variation in the RALDH2 locus and congenital heart disease. Six different SNPs were analyzed in 101 patient-parents trios in a TDT study. None of the markers displayed any association with CHD. No single haplotype was associated with an increased risk of CHD. Analysis of the A151G polymorphism indicated that the variant produced substantial changes in mRNA structure. This variant is also localized in a putative exonic splicing enhancer (ESE). Functional splicing studies failed to reveal a significant impact of this variant and gene splicing. This methodology was applied to another mutation (G151T) found in exon 4 during the sequencing of RALDH2 gene and an increase in splicing signal was observed. We found four mutations more: rs34645259, T157G (exon 4), rs4646626 and rs35251510. In summary, no association between CHD and genetic variation at the RALDH2 locus in humans was found. Potential functional genetic variants should be further studied in order to define their real role in RA pathway disturbances

Transtorno de déficit de atenção e hiperatividade: um olhar no ensino fundamental

Nos últimos anos, o diagnóstico de TDAH aumentou no contexto escolar, aumentando também a quantidade de pesquisas sobre o assunto. O presente estudo investigou o número de alunos com TDAH em 17 escolas de Porto Alegre e a percepção de 136 professores de 1ª à 8ª série, sobre o transtorno e o comportamento das crianças com TDAH. Dois questionários foram utilizados: um preenchido pela direção da escola e o outro, pelos professores. A média dos alunos com TDAH das escolas estudadas foi de 3%, com dois casos extremos: uma escola com 51% e outra com 0,2%. Em relação à percepção dos professores,houve discrepância entre os possíveis casos apontados por estes e os levantados junto à direção. Conclui- se, portanto, que é necessário oferecer ao ambiente escolar mais informação sobre esse transtorno, pois a maioria das escolas estudadas não oferece subsídios aos professores.In the last years the Attention Deficit Hyperactive Disorder (ADHD) has increased in schools. This study investigates the number of students reported with the ADHD in 17 schools in Porto Alegre, a city in the south of Brazil. One hundred and thirty six 1st the to 8 grade teachers were asked about their perceptions concerning this behavioral disorder. Two questionnaires filled in by the school direction and by the teachers, respectively, were used. The average of students with ADHD in the schools was 3% - one school reported 51% and the other 0.2%. Concerning teachers’ perceptions it was found a discrepancy between cases identified as ADHD by the teachers and the cases reported by the school direction. It is necessary to offer more information to the schools on the ADHD since most of them do not provide subsidies for the teachers regarding this matter

Attention deficit hyperactivity disorder: a glance at the elementary school

Submitted by Bruna Vieira ([email protected]) on 2011-11-02T18:46:47Z No. of bitstreams: 1 Transtorno de déficti de atenção e hiperatividade um olhar no ensino fundamental.pdf: 573392 bytes, checksum: 9559da5ea6ba1a3d1559589d4ea9ab27 (MD5)Approved for entry into archive by Barbara Milbrath([email protected]) on 2011-11-04T10:16:41Z (GMT) No. of bitstreams: 1 Transtorno de déficti de atenção e hiperatividade um olhar no ensino fundamental.pdf: 573392 bytes, checksum: 9559da5ea6ba1a3d1559589d4ea9ab27 (MD5)Made available in DSpace on 2011-11-04T10:16:41Z (GMT). No. of bitstreams: 1 Transtorno de déficti de atenção e hiperatividade um olhar no ensino fundamental.pdf: 573392 bytes, checksum: 9559da5ea6ba1a3d1559589d4ea9ab27 (MD5) Previous issue date: 2008Nos últimos anos, o diagnóstico de TDAH aumentou no contexto escolar, aumentando também a quantidade de pesquisas sobre o assunto. O presente estudo investigou o número de alunos com TDAH em 17 escolas de Porto Alegre e a percepção de 136 professores de 1ª à 8ª série, sobre o transtorno e o comportamento das crianças com TDAH. Dois questionários foram utilizados: um preenchido pela direção da escola e o outro, pelos professores. A média dos alunos com TDAH das escolas estudadas foi de 3%, com dois casos extremos: uma escola com 51% e outra com 0,2%. Em relação à percepção dos professores,houve discrepância entre os possíveis casos apontados por estes e os levantados junto à direção. Conclui- se, portanto, que é necessário oferecer ao ambiente escolar mais informação sobre esse transtorno, pois a maioria das escolas estudadas não oferece subsídios aos professores.In the last years the Attention Deficit Hyperactive Disorder (ADHD) has increased in schools. This study investigates the number of students reported with the ADHD in 17 schools in Porto Alegre, a city in the south of Brazil. One hundred and thirty six 1st the to 8 grade teachers were asked about their perceptions concerning this behavioral disorder. Two questionnaires filled in by the school direction and by the teachers, respectively, were used. The average of students with ADHD in the schools was 3% - one school reported 51% and the other 0.2%. Concerning teachers’ perceptions it was found a discrepancy between cases identified as ADHD by the teachers and the cases reported by the school direction. It is necessary to offer more information to the schools on the ADHD since most of them do not provide subsidies for the teachers regarding this matter