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Björk, S., Schappert, M. & Jönsson, H. (2020). Droplet microfluidic microcolony sorting by fluorescence area for high throughput, yield-based screening of triacyl glycerides in S. Cerevisiae. In: MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences: . Paper presented at 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2020, 4 October 2020 through 9 October 2020 (pp. 1015-1016). Chemical and Biological Microsystems Society
Open this publication in new window or tab >>Droplet microfluidic microcolony sorting by fluorescence area for high throughput, yield-based screening of triacyl glycerides in S. Cerevisiae
2020 (English)In: MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Chemical and Biological Microsystems Society , 2020, p. 1015-1016Conference paper, Published paper (Refereed)
Abstract [en]

Here we present a droplet microfluidics workflow for cell factory screening by yield of an intracellular product from isogenic microcolonies, i.e. minimal cell populations, encapsulated in picoliter droplets. This allows us to utilize all the benefits of droplet microfluidic screening in terms of throughput, but based on the signal from a population average, rather than the noisy single cell signal. We demonstrate microcolony sorting by integrated droplet fluorescence area of encapsulated E. coli, optimize triglyceride (TG) microcolony assay in droplets and apply the microcolony screening concept to analyze triglyceride (TG) production in S. cerevisiae.

Place, publisher, year, edition, pages
Chemical and Biological Microsystems Society, 2020
Keywords
Droplet microfluidics, High throughput screening, Yeast cell factories, Cell culture, Cell proliferation, Drops, Escherichia coli, Fluorescence, Cell populations, High throughput, Intracellular products, Microcolonies, Picoliter droplets, Single cells, Triacyl glyceride, Microfluidics
National Category
Biochemistry Molecular Biology
Identifiers
urn:nbn:se:kth:diva-302926 (URN)2-s2.0-85098249928 (Scopus ID)
Conference
24th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2020, 4 October 2020 through 9 October 2020
Note

QC 20211001

Available from: 2021-10-01 Created: 2021-10-01 Last updated: 2025-02-20Bibliographically approved
Yao, L., Shabestary, K., Björk, S. M., Asplund-Samuelsson, J., Joensson, H. N., Jahn, M. & Hudson, E. P. (2020). Pooled CRISPRi screening of the cyanobacterium Synechocystis sp PCC 6803 for enhanced industrial phenotypes. Nature Communications, 11(1), Article ID 1666.
Open this publication in new window or tab >>Pooled CRISPRi screening of the cyanobacterium Synechocystis sp PCC 6803 for enhanced industrial phenotypes
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2020 (English)In: Nature Communications, E-ISSN 2041-1723, Vol. 11, no 1, article id 1666Article in journal (Refereed) Published
Abstract [en]

Cyanobacteria are model organisms for photosynthesis and are attractive for biotechnology applications. To aid investigation of genotype-phenotype relationships in cyanobacteria, we develop an inducible CRISPRi gene repression library in Synechocystis sp. PCC 6803, where we aim to target all genes for repression. We track the growth of all library members in multiple conditions and estimate gene fitness. The library reveals several clones with increased growth rates, and these have a common upregulation of genes related to cyclic electron flow. We challenge the library with 0.1 M L-lactate and find that repression of peroxiredoxin bcp2 increases growth rate by 49%. Transforming the library into an L-lactate-secreting Synechocystis strain and sorting top lactate producers enriches clones with sgRNAs targeting nutrient assimilation, central carbon metabolism, and cyclic electron flow. In many examples, productivity can be enhanced by repression of essential genes, which are difficult to access by transposon insertion.

Place, publisher, year, edition, pages
Nature Research, 2020
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-276263 (URN)10.1038/s41467-020-15491-7 (DOI)000564272800006 ()32245970 (PubMedID)2-s2.0-85083041505 (Scopus ID)
Note

QC 20200622

Available from: 2020-06-22 Created: 2020-06-22 Last updated: 2024-03-18Bibliographically approved
Björk, S. & Jönsson, H. (2019). Microfluidics for cell factory and bioprocess development. Current Opinion in Biotechnology, 55, 95-102
Open this publication in new window or tab >>Microfluidics for cell factory and bioprocess development
2019 (English)In: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 55, p. 95-102Article in journal (Refereed) Published
Abstract [en]

Bioindustry is expanding to an increasing variety of food, chemical and pharmaceutical products, each requiring rapid development of a dedicated cell factory and bioprocess. Microfluidic tools are, together with tools from synthetic biology and metabolic modeling, being employed in cell factory and bioprocess development to speed up development and address new products. Recent examples of microfluidics for bioprocess development range from integrated devices for DNA assembly and transformation, to high throughput screening of cell factory libraries, and micron scale bioreactors for process optimization. These improvements act to improve the biotechnological engineering cycle with tools for building, testing and evaluating cell factories and bioprocesses by increasing throughput, parallelization and automation.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Cells, Cytology, Drug products, Microfluidics, Optimization, Bioprocess development, DNA assemblies, High throughput screening, Integrated device, Metabolic modeling, Parallelizations, Pharmaceutical products, Synthetic biology, Cell engineering, bioprocess, biotechnological procedures, cell factory, cell growth, cell population, cell selection, cell separation, priority journal, process optimization, Review, single cell analysis
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-236324 (URN)10.1016/j.copbio.2018.08.011 (DOI)000459949400015 ()30236890 (PubMedID)2-s2.0-85053411311 (Scopus ID)
Funder
Novo NordiskKnut and Alice Wallenberg FoundationSwedish Research Council Formas
Note

QC 20181120

Available from: 2018-11-20 Created: 2018-11-20 Last updated: 2022-06-26Bibliographically approved
Wang, G., Björk, S., Huang, M., Liu, Q., Campbell, K., Nielsen, J., . . . Petranovic, D. (2019). RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences of the United States of America, 116(19), 9324-9332
Open this publication in new window or tab >>RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae
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2019 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 116, no 19, p. 9324-9332Article in journal (Refereed) Published
Abstract [en]

The cellular machinery that supports protein synthesis and secretion lies at the foundation of cell factory-centered protein production. Due to the complexity of such cellular machinery, the challenge in generating a superior cell factory is to fully exploit the production potential by finding beneficial targets for optimized strains, which ideally could be used for improved secretion of other proteins. We focused on an approach in the yeast Saccharomyces cerevisiae that allows for attenuation of gene expression, using RNAi combined with high-throughput microfluidic single-cell screening for cells with improved protein secretion. Using direct experimental validation or enrichment analysis-assisted characterization of systematically introduced RNAi perturbations, we could identify targets that improve protein secretion. We found that genes with functions in cellular metabolism (YDC1, AAD4, ADE8, and SDH1), protein modification and degradation (VPS73, KTR2, CNL1, and SSA1), and cell cycle (CDC39), can all impact recombinant protein production when expressed at differentially down regulated levels. By establishing a workflow that incorporates Cas9-mediated recombineering, we demonstrated how we could tune the expression of the identified gene targets for further improved protein production for specific proteins. Our findings offer a high throughput and semirational platform design, which will improve not only the production of a desired protein but even more importantly, shed additional light on connections between protein production and other cellular processes.

Place, publisher, year, edition, pages
National Academy of Sciences, 2019
National Category
Industrial Biotechnology Biochemistry Molecular Biology Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-250438 (URN)10.1073/pnas.1820561116 (DOI)000467226400031 ()31000602 (PubMedID)2-s2.0-85065621578 (Scopus ID)
Note

QC 20190529

Available from: 2019-04-29 Created: 2019-04-29 Last updated: 2025-02-20Bibliographically approved
Björk, S. M., Sjostrom, S. L., Andersson-Svahn, H. & Jönsson, H. N. (2015). Metabolite profiling of microfluidic cell culture conditions for droplet based screening. Biomicrofluidics, 9(4), Article ID 044128.
Open this publication in new window or tab >>Metabolite profiling of microfluidic cell culture conditions for droplet based screening
2015 (English)In: Biomicrofluidics, E-ISSN 1932-1058, Vol. 9, no 4, article id 044128Article in journal (Refereed) Published
Abstract [en]

We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell/clone screening in droplets, such as directed evolution of yeast, as cell metabolic state directly affects production yields from cell factories. Here, we analyze glucose, pyruvate, ethanol, and glycerol, central metabolites in yeast glucose dissimilation to establish culture formats for screening of respiring as well as fermenting yeast. Metabolite profiling provides a more nuanced estimate of cell state compared to proliferation studies alone. We show that the choice of droplet incubation format impacts cell proliferation and metabolite production. The standard syringe incubation of droplets exhibited metabolite profiles similar to oxygen limited cultures, whereas the metabolite profiles of cells cultured in the alternative wide tube droplet incubation format resemble those from aerobic culture. Furthermore, we demonstrate retained droplet stability and size in the new better oxygenated droplet incubation format.

Keywords
Biomolecules;Cell culture;Cell proliferation;Cells;Dimensional stability;Glucose;Metabolism;Metabolites;Microfluidics;Yeast
National Category
Biochemistry Molecular Biology
Identifiers
urn:nbn:se:kth:diva-173782 (URN)10.1063/1.4929520 (DOI)000360311900030 ()26392830 (PubMedID)2-s2.0-84940909670 (Scopus ID)
Funder
Science for Life Laboratory, SciLifeLab
Note

QC 20150921

QC 20191008

Available from: 2015-09-21 Created: 2015-09-18 Last updated: 2025-02-20Bibliographically approved
Björk, S. M., Sjöström, S. L., Andersson-Svahn, H. & Jönsson, H. N. (2015). Tuning microfluidic cell culture conditions for droplet based screening by metabolite profiling. In: MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences: . Paper presented at 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2015, 25 October 2015 through 29 October 2015 (pp. 1377-1379). Chemical and Biological Microsystems Society
Open this publication in new window or tab >>Tuning microfluidic cell culture conditions for droplet based screening by metabolite profiling
2015 (English)In: MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Chemical and Biological Microsystems Society , 2015, p. 1377-1379Conference paper, Published paper (Refereed)
Abstract [en]

We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell screening in droplets, as cell metabolic state directly affects production yields in cell factories. Metabolite profiling provides a more nuanced estimate of cell state compared to proliferation studies alone. We show that the choice of droplet incubation format has an impact on cell proliferation and metabolite production. Furthermore, we engineered a new better oxygenated droplet incubation format, with retained droplet stability and size.

Place, publisher, year, edition, pages
Chemical and Biological Microsystems Society, 2015
Keywords
Cell culture, Cell metabolism, Droplet microfluidics, Biomolecules, Cell proliferation, Cells, Dimensional stability, Drops, Metabolism, Metabolites, Microfluidics, Culture conditions, Metabolite concentrations, Metabolite production, Metabolite profiling, Microfluidic cell culture, Microfluidic droplets, Cytology
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-194643 (URN)2-s2.0-84983371580 (Scopus ID)9780979806483 (ISBN)
Conference
19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2015, 25 October 2015 through 29 October 2015
Note

QC 20161129

Available from: 2016-11-29 Created: 2016-10-31 Last updated: 2024-03-15Bibliographically approved
Björk, S., Sjöström, S. L., Andersson Svahn, H. & Jönsson, H. N. Controlling cell metabolic state in droplet microfluidics.
Open this publication in new window or tab >>Controlling cell metabolic state in droplet microfluidics
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry Molecular Biology
Identifiers
urn:nbn:se:kth:diva-159308 (URN)
Note

QS 2015

Available from: 2015-01-29 Created: 2015-01-29 Last updated: 2025-02-20Bibliographically approved
Björk, S., Schappert, M. & Jönsson, H.Droplet microfluidic microcolony analysis of triacylglycerol yields in S. cerevisiae for high throughput screening.
Open this publication in new window or tab >>Droplet microfluidic microcolony analysis of triacylglycerol yields in S. cerevisiae for high throughput screening
(English)Manuscript (preprint) (Other academic)
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-259488 (URN)
Available from: 2019-09-16 Created: 2019-09-16 Last updated: 2022-06-26Bibliographically approved
Björk, S., Shabestary, K., Yao, L., Ljungqvist, E., Jönsson, H. & Hudson, E. P.Droplet microfluidic screening of a Synechocystis sp. CRISPRi library based on L-lactate production.
Open this publication in new window or tab >>Droplet microfluidic screening of a Synechocystis sp. CRISPRi library based on L-lactate production
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(English)Manuscript (preprint) (Other academic)
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-259487 (URN)
Note

QC 20191011

Available from: 2019-09-16 Created: 2019-09-16 Last updated: 2022-06-26Bibliographically approved
Trossbach, M., Björk, S. & Jönsson, H.High-throughput fluorescence area sorting of droplet microfluidic S. cerevisiae microcolonies.
Open this publication in new window or tab >>High-throughput fluorescence area sorting of droplet microfluidic S. cerevisiae microcolonies
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Cellular heterogeneity in isogenic cell populations is a major obstacle for single-cell screening campaigns, as the phenotype of individual cells might differ drastically from the mean, leading to large overlaps between productivity assessments of populations. At the other end of the spectrum, isogenic bulk assays provide a more accurate picture of a strain’s capacity at production scale, but suffers from low throughput and high reagent consumption.

Here, we present a screening format for cell factory variant libraries, aiming at combining the advantages of single-cell screening and bulk assay formats. Using microfluidic droplets, we compartmentalize yeast cell producer candidates, culture them to form isogenic microcolonies and sort colonies at higher throughput than bulk experiments to assess the genetic potential more accurately than in a single-cell screening format. To this end, we developed a fluorescence area-based sorting method that integrates the fluorescence signal from the entire fluorescence profile of a droplet and bases the sorting decision on that integrated fluorescence area. We validate the concept by sorting droplet microcolonies of fluorescent protein expressing Escherichia coli. Finally, we successfully sorted encapsulated iso-genic microcolonies of a low-producing and a high-producing strain of Saccharomyces cerevisiae by Triacylglycerol (TAG) production at 220 Hz, enriching the high-producing strain 4.45-fold.

National Category
Bioenergy
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-321635 (URN)
Note

QC 20221129

Available from: 2022-11-18 Created: 2022-11-18 Last updated: 2022-11-29Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-3722-5970

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