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      A Multi-Platform Flow Device for Microbial (Co-) Cultivation and Microscopic Analysis

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          Abstract

          Novel microbial cultivation platforms are of increasing interest to researchers in academia and industry. The development of materials with specialized chemical and geometric properties has opened up new possibilities in the study of previously unculturable microorganisms and has facilitated the design of elegant, high-throughput experimental set-ups. Within the context of the international Genetically Engineered Machine (iGEM) competition, we set out to design, manufacture, and implement a flow device that can accommodate multiple growth platforms, that is, a silicon nitride based microsieve and a porous aluminium oxide based microdish. It provides control over (co-)culturing conditions similar to a chemostat, while allowing organisms to be observed microscopically. The device was designed to be affordable, reusable, and above all, versatile. To test its functionality and general utility, we performed multiple experiments with Escherichia coli cells harboring synthetic gene circuits and were able to quantitatively study emerging expression dynamics in real-time via fluorescence microscopy. Furthermore, we demonstrated that the device provides a unique environment for the cultivation of nematodes, suggesting that the device could also prove useful in microscopy studies of multicellular microorganisms.

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          Most cited references30

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          Engineering BioBrick vectors from BioBrick parts

          Background The underlying goal of synthetic biology is to make the process of engineering biological systems easier. Recent work has focused on defining and developing standard biological parts. The technical standard that has gained the most traction in the synthetic biology community is the BioBrick standard for physical composition of genetic parts. Parts that conform to the BioBrick assembly standard are BioBrick standard biological parts. To date, over 2,000 BioBrick parts have been contributed to, and are available from, the Registry of Standard Biological Parts. Results Here we extended the same advantages of BioBrick standard biological parts to the plasmid-based vectors that are used to provide and propagate BioBrick parts. We developed a process for engineering BioBrick vectors from BioBrick parts. We designed a new set of BioBrick parts that encode many useful vector functions. We combined the new parts to make a BioBrick base vector that facilitates BioBrick vector construction. We demonstrated the utility of the process by constructing seven new BioBrick vectors. We also successfully used the resulting vectors to assemble and propagate other BioBrick standard biological parts. Conclusion We extended the principles of part reuse and standardization to BioBrick vectors. As a result, myriad new BioBrick vectors can be readily produced from all existing and newly designed BioBrick parts. We invite the synthetic biology community to (1) use the process to make and share new BioBrick vectors; (2) expand the current collection of BioBrick vector parts; and (3) characterize and improve the available collection of BioBrick vector parts.
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            Cultivating the uncultured.

            The recent application of molecular phylogeny to environmental samples has resulted in the discovery of an abundance of unique and previously unrecognized microorganisms. The vast majority of this microbial diversity has proved refractory to cultivation. Here, we describe a universal method that provides access to this immense reservoir of untapped microbial diversity. This technique combines encapsulation of cells in gel microdroplets for massively parallel microbial cultivation under low nutrient flux conditions, followed by flow cytometry to detect microdroplets containing microcolonies. The ability to grow and study previously uncultured organisms in pure culture will enhance our understanding of microbial physiology and metabolic adaptation and will provide new sources of microbial metabolites. We show that this technology can be applied to samples from several different environments, including seawater and soil.
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              Long-term monitoring of bacteria undergoing programmed population control in a microchemostat.

              Using an active approach to preventing biofilm formation, we implemented a microfluidic bioreactor that enables long-term culture and monitoring of extremely small populations of bacteria with single-cell resolution. We used this device to observe the dynamics of Escherichia coli carrying a synthetic "population control" circuit that regulates cell density through a feedback mechanism based on quorum sensing. The microfluidic bioreactor enabled long-term monitoring of unnatural behavior programmed by the synthetic circuit, which included sustained oscillations in cell density and associated morphological changes, over hundreds of hours.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                14 May 2012
                : 7
                : 5
                : e36982
                Affiliations
                [1 ]Wageningen UR iGEM 2011 Team, Wageningen University, Wageningen, The Netherlands
                [2 ]MicroDish BV, Utrecht, The Netherlands
                [3 ]Food Process Engineering, Wageningen University, Wageningen, The Netherlands
                [4 ]Systems and Synthetic Biology, Wageningen University, Wageningen, The Netherlands
                [5 ]Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands
                [6 ]Netherlands Consortium for Systems Biology, University of Amsterdam, Amsterdam, The Netherlands
                University of Hyderabad, India
                Author notes

                Conceived and designed the experiments: MCH DIO BMR FH MWJvP. Performed the experiments: MCH DIO BMR. Analyzed the data: MCH DIO BMR SdG RvH JK PK DN YvN ES RS HdV MTW KS CJI VAPMdS SKS FH MWJvP. Contributed reagents/materials/analysis tools: CJI KS. Wrote the paper: MCH DIO BMR SdG RvH JK PK DN YvN ES RS HdV MTW CJI KS VAPMdS SKS FH MWJvP.

                Article
                PONE-D-11-23621
                10.1371/journal.pone.0036982
                3351485
                22606321
                4c57fb1d-267d-43a4-b6ff-5669bb073012
                Hesselman 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
                : 24 November 2011
                : 11 April 2012
                Page count
                Pages: 8
                Categories
                Research Article
                Biology
                Biotechnology
                Applied Microbiology
                Developmental Biology
                Microbial Growth and Development
                Ecology
                Microbial Ecology
                Microbiology
                Applied Microbiology
                Industrial Microbiology
                Microbial Ecology
                Microbial Growth and Development

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                Uncategorized

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