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      Low cost and open source multi-fluorescence imaging system for teaching and research in biology and bioengineering

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          Abstract

          The advent of easy-to-use open source microcontrollers, off-the-shelf electronics and customizable manufacturing technologies has facilitated the development of inexpensive scientific devices and laboratory equipment. In this study, we describe an imaging system that integrates low-cost and open-source hardware, software and genetic resources. The multi-fluorescence imaging system consists of readily available 470 nm LEDs, a Raspberry Pi camera and a set of filters made with low cost acrylics. This device allows imaging in scales ranging from single colonies to entire plates. We developed a set of genetic components (e.g. promoters, coding sequences, terminators) and vectors following the standard framework of Golden Gate, which allowed the fabrication of genetic constructs in a combinatorial, low cost and robust manner. In order to provide simultaneous imaging of multiple wavelength signals, we screened a series of long stokes shift fluorescent proteins that could be combined with cyan/green fluorescent proteins. We found CyOFP1, mBeRFP and sfGFP to be the most compatible set for 3-channel fluorescent imaging. We developed open source Python code to operate the hardware to run time-lapse experiments with automated control of illumination and camera and a Python module to analyze data and extract meaningful biological information. To demonstrate the potential application of this integral system, we tested its performance on a diverse range of imaging assays often used in disciplines such as microbial ecology, microbiology and synthetic biology. We also assessed its potential use in a high school environment to teach biology, hardware design, optics, and programming. Together, these results demonstrate the successful integration of open source hardware, software, genetic resources and customizable manufacturing to obtain a powerful, low cost and robust system for education, scientific research and bioengineering. All the resources developed here are available under open source licenses.

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          Most cited references43

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          Engineering and characterization of a superfolder green fluorescent protein.

          Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP.
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            Improving FRET dynamic range with bright green and red fluorescent proteins

            A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of cyan and yellow fluorescent proteins (CFP and YFP), but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light, and complex photokinetic events such as reversible photobleaching and photoconversion. Here, we engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date, and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP in reporters of kinase activity, small GTPase activity, and transmembrane voltage significantly improves photostability, FRET dynamic range, and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action potential firing and RhoA activation in growth cones.
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              Genetic drift at expanding frontiers promotes gene segregation.

              Competition between random genetic drift and natural selection play a central role in evolution: Whereas nonbeneficial mutations often prevail in small populations by chance, mutations that sweep through large populations typically confer a selective advantage. Here, however, we observe chance effects during range expansions that dramatically alter the gene pool even in large microbial populations. Initially well mixed populations of two fluorescently labeled strains of Escherichia coli develop well defined, sector-like regions with fractal boundaries in expanding colonies. The formation of these regions is driven by random fluctuations that originate in a thin band of pioneers at the expanding frontier. A comparison of bacterial and yeast colonies (Saccharomyces cerevisiae) suggests that this large-scale genetic sectoring is a generic phenomenon that may provide a detectable footprint of past range expansions.
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                Author and article information

                Contributors
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: SoftwareRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ResourcesRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: ResourcesRole: SoftwareRole: Writing – original draft
                Role: ConceptualizationRole: Funding acquisitionRole: Validation
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: Project administrationRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: Project administrationRole: SupervisionRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                15 November 2017
                2017
                : 12
                : 11
                : e0187163
                Affiliations
                [1 ] Department of Chemical and Bioprocess Engineering, School of Engineering, Pontificia Universidad Católica de Chile, Santiago, Chile
                [2 ] Backyard Brains, Santiago, Chile
                [3 ] Departamento Ecología, Facultad Ciencias Biológicas; Instituto de Física, Facultad de Física, Pontificia Universidad Católica de Chile, Santiago, Chile
                [4 ] Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, Chile
                [5 ] Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile
                [6 ] Fondo de Desarrollo de Áreas Prioritarias, Center for Genome Regulation, Millennium Nucleus Center for Plant Systems and Synthetic Biology, Pontificia Universidad Católica de Chile, Santiago, Chile
                Imperial College London, UNITED KINGDOM
                Author notes

                Competing Interests: Authors TCM and RHP contributed to this work under employment by Backyard Brains, a company that develops and distributes open source scientific equipment. Backyard Brains worked on this project under support of the OpenPlant fund. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Additionally, there are no patents, products in development or marketed products to declare.

                Author information
                http://orcid.org/0000-0001-9200-5383
                Article
                PONE-D-17-30866
                10.1371/journal.pone.0187163
                5687719
                29140977
                29dd63d0-db2c-4b83-9f5f-af00412129fb
                © 2017 Nuñez et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 22 August 2017
                : 14 October 2017
                Page count
                Figures: 6, Tables: 1, Pages: 21
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100002848, Comisión Nacional de Investigación Científica y Tecnológica;
                Award ID: 11140776
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100002848, Comisión Nacional de Investigación Científica y Tecnológica;
                Award ID: 11161046
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100002848, Comisión Nacional de Investigación Científica y Tecnológica;
                Award ID: 1150430
                Award Recipient :
                Funded by: Comisión Nacional de Investigación Científica y Tecnológica (CL)
                Award ID: 15090007
                Funded by: Ministerio de Economía, Fomento y Turismo (CL)
                Award ID: NC130030
                Funded by: OpenPlant Fund
                Award Recipient :
                We would like to thank the funders of this study: OpenPlant Fund for financial support to INN, TFM, RHP, JEK, TCM, TJR, and FF; CONICYT Fondecyt Iniciacion 11140776 for providing financial support to FF; CONICYT Fondecyt Iniciación 11161046 for providing support to TJR; Fondo de Desarrollo de Areas Prioritarias (FONDAP) Center for Genome Regulation (15090007) and Millennium Nucleus Center for Plant Systems and Synthetic Biology (NC130030) for providing financial support to FF; CONICYT Fondecyt Regular 1150430 for providing financial support to JEK and Backyard Brains for providing support in the form of salaries for TCM and RHP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.
                Categories
                Research Article
                Research and Analysis Methods
                Imaging Techniques
                Fluorescence Imaging
                Engineering and Technology
                Equipment
                Optical Equipment
                Cameras
                Computer and Information Sciences
                Computer Software
                Open Source Software
                Science Policy
                Open Science
                Open Source Software
                Engineering and Technology
                Signal Processing
                Signal Filtering
                Biology and Life Sciences
                Biochemistry
                Ribosomes
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Ribosomes
                Engineering and Technology
                Equipment
                Imaging Equipment
                Research and analysis methods
                Chemical characterization
                Binding analysis
                DNA binding assay
                Engineering and Technology
                Equipment
                Open Source Hardware
                Custom metadata
                All sequence and images files are available from the Open Science framework database ( https://osf.io/dy6p2/). All the documentation for hardware is available at Docubricks ( http://docubricks.com/viewer.jsp?id=701517893260717056). Code is available from GitHUB ( www.github.com/synbiouc/fluopi).

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