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. 2018 Feb 14;8:3000. doi: 10.1038/s41598-018-20877-1

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Enrichment of high B₂ producing strains by buoyancy separation. (a) Principle of the screen: The nLRs are inoculated with B₂ producing (orange) and B₂ sensing (green) E. coli cells. The sensing cells take up B₂ via RibM and convert it to FMN. FMN then translationally upregulates synthesis of a Cat-GFP fusion protein, thereby providing the basis for two-fold identification of potent B₂ secreting isolates. (b) Upper panel: E. coli BW23474 whole cell biocatalysts were equipped with one of four different plasmids leading to different degrees of B₂ overproduction in shake flasks (lower panel, orange columns). Next, the biocatalysts were encapsulated in nLRs (on average 0.2 cells per nLR) together with biosensors (on average 1,000 cells per nLR). The nLRs were incubated, subjected to separation on a microscope slide in a water droplet, and the buoyancy-positive nLRs were counted under the microscope and analysed for GFP fluorescence. Only the high B₂ producing strain led to a large fraction of ascending nLRs (pB2_ribDBECA: 114 of 120), whereas the others did not (pB2_ribDBECA_PL: 0 of 60; pB2_ribBECA: 0 of 57; pB2_empty: 3 of 101). One-way ANOVA indicated a statistically significant difference among the different strains for B₂ secretion (F(2,15) = 1312.57, p < 0.00001, α = 0.01) and separation (F(3,14) = 1463.94, p < 0.00001, α = 0.01). Data shown as mean ± SD, n = 4 to 5 with 11 to 25 nLRs per experiment. (c) Approx. 363,000 nLRs were inoculated with approx. 79,000 E. coli cells obtained from four different B₂ producing whole cell biocatalysts (see b). The most potent B₂ producing whole cell biocatalyst carrying plasmid pB2_ribDBECA was underrepresented (660 cells, 0.8%). An overlay of bright field and epifluorescence microscopic images of the nLR population before (left image, bulk) and after (right picture, top fraction) buoyancy separation indicates efficient enrichment of nLRs with Cat-GFP overexpressing biosensors. These observations were verified by counting the fluorescently labelled high B₂-producer containing nLRs within the total population by large-particle flow cytometry (549 nLRs). Scale bar: 500 µm.