Dataset Information

Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli.

ABSTRACT: We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices.

SUBMITTER: Beal J

PROVIDER: S-EPMC4777433 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli.

Beal Jacob J Haddock-Angelli Traci T Gershater Markus M de Mora Kim K Lizarazo Meagan M Hollenhorst Jim J Rettberg Randy R

PloS one 20160303 3

We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54- ...[more]

PMID: 26937966

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Project description:Biological approaches to intervertebral disc (IVD) restoration and/or regeneration have become of increasing interest. However, the IVD comprises a viscoelastic system whose biological replacement remains challenging. The present study sought to design load-sharing two-component model systems of circular, nested, concentric elements reflecting the nucleus pulposus and annulus fibrosus. Specifically, we wanted to investigate the effect of architectural design variations on (1) model system failure loads when testing the individual materials either separately or homogeneously mixed, and (2) also evaluate the potential of modulating other mechanical properties of the model systems. Two sets of softer and harder biomaterials, 0.5% and 5% agarose vs. 0.5% agarose and gelatin, were used for fabrication. Architectural design variations were realized by varying ring geometries and amounts while keeping the material composition across designs comparable. Variations in the architectural design, such as lamellar width, number, and order, combined with choosing specific biomaterial properties, strongly influenced the biomechanical performance of IVD constructs. Biomechanical characterization revealed that the single most important parameter, in which the model systems vastly exceeded those of the individual materials, was failure load. The model system failure loads were 32.21- and 84.11-fold higher than those of the agarose materials and 55.03- and 2.14-fold higher than those of the agarose and gelatin materials used for system fabrication. The compressive strength, dynamic stiffness, and viscoelasticity of the model systems were always in the range of the individual materials. Relevant architecture-promoted biomechanical performance-tuning of tissue-engineered constructs for biological IVD replacement can be realized by slight modifications in the design of constructs while preserving the materials' compositions. Minimal variations in the architectural design can be used to precisely control structure-function relations for IVD constructs rather than choosing different materials. These fundamental findings have important implications for efficient tissue-engineering of IVDs and other load-bearing tissues, as potential implants need to withstand high in situ loads.

Project description:BackgroundMolecular mechanisms generating genetic variation provide the basis for evolution and long-term survival of a population in a changing environment. In stable, laboratory conditions, the variation-generating mechanisms are dispensable, as there is limited need for the cell to adapt to adverse conditions. In fact, newly emerging, evolved features might be undesirable when working on highly refined, precise molecular and synthetic biological tasks.ResultsBy constructing low-mutation-rate variants, we reduced the evolutionary capacity of MDS42, a reduced-genome E. coli strain engineered to lack most genes irrelevant for laboratory/industrial applications. Elimination of diversity-generating, error-prone DNA polymerase enzymes involved in induced mutagenesis achieved a significant stabilization of the genome. The resulting strain, while retaining normal growth, showed a significant decrease in overall mutation rates, most notably under various stress conditions. Moreover, the error-prone polymerase-free host allowed relatively stable maintenance of a toxic methyltransferase-expressing clone. In contrast, the parental strain produced mutant clones, unable to produce functional methyltransferase, which quickly overgrew the culture to a high ratio (50% of clones in a 24-h induction period lacked functional methyltransferase activity). The surprisingly large stability-difference observed between the strains was due to the combined effects of high stress-induced mutagenesis in the parental strain, growth inhibition by expression of the toxic protein, and selection/outgrowth of mutants no longer producing an active, toxic enzyme.ConclusionsBy eliminating stress-inducible error-prone DNA-polymerases, the genome of the mobile genetic element-free E. coli strain MDS42 was further stabilized. The resulting strain represents an improved host in various synthetic and molecular biological applications, allowing more stable production of growth-inhibiting biomolecules.

Dataset Information

Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli.

Publications

Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli.

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