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Droplet microfluidics for screening and sorting of microbial cell factories
KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi. (Nanobiotechnology)
2019 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Cell factories are cells that have been engineered to produce a compound of interest, ranging from biopharmaceuticals to biofuels. With advances in metabolic engineering, the number of cell factory variants to evaluate has increased dramatically, necessitating screening methods with increased throughput. Microfluidic droplets, which can be generated, manipulated and interrogated at very high throughput, are isolated reaction vessels at the single cell scale. Compartmentalization maintains the genotype-phenotype link, making droplet microfluidics suitable for screening of extracellular traits such as secreted products and for screening of microcolonies originating from single cells.

 

In Paper I, we investigated the impact of droplet microfluidic incubation formats on cell culture conditions and found that syringe and semi open incubation resulted in different metabolic profiles. Controlling culture conditions is key to cell factory screening, as product formation is influenced by the state of the cell.

 

In Paper II and III, we used droplet microfluidics to perform screening campaigns of interference based cell factory variant libraries. In Paper II, two S. cerevisiae RNAi libraries were screened based on amylase secretion, and from the sorted fraction genes linked to improved protein secretion could be identified. In paper III, we screened a Synecosystis sp. CRISPRi library based on lactate secretion. The library was sorted at different time points after induction, followed by sequencing to reveal genes enriched by droplet sorting.

 

In Paper IV, we developed a droplet microcolony-based assay for screening intracellular triacylglycerol (TAG) in S. cerevisiae, and showed improved strain separation compared to flow cytometry in a hypothetical sorting scenario. By screening microcolonies compartmentalized in droplets, we combine the throughput of single cell screening methods with the reduced impact of cell-to-cell noise in cell ensemble analysis.

sted, utgiver, år, opplag, sider
KTH Royal Institute of Technology, 2019. , s. 58
Serie
TRITA-CBH-FOU ; 2019:43
Emneord [en]
Droplet microfluidics, Cell factories, High-throughput screening, Cell culture
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
URN: urn:nbn:se:kth:diva-259490ISBN: 978-91-7873-290-6 (tryckt)OAI: oai:DiVA.org:kth-259490DiVA, id: diva2:1351561
Disputas
2019-10-11, Air & Fire, Tomtebodavägen 23A, Solna, 10:00 (engelsk)
Opponent
Veileder
Merknad

QC 2019-09-16

Tilgjengelig fra: 2019-09-16 Laget: 2019-09-16 Sist oppdatert: 2019-09-16bibliografisk kontrollert
Delarbeid
1. Metabolite profiling of microfluidic cell culture conditions for droplet based screening
Åpne denne publikasjonen i ny fane eller vindu >>Metabolite profiling of microfluidic cell culture conditions for droplet based screening
2015 (engelsk)Inngår i: Biomicrofluidics, ISSN 1932-1058, E-ISSN 1932-1058, Vol. 9, nr 4, artikkel-id 044128Artikkel i tidsskrift (Fagfellevurdert) 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.

Emneord
Biomolecules;Cell culture;Cell proliferation;Cells;Dimensional stability;Glucose;Metabolism;Metabolites;Microfluidics;Yeast
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-173782 (URN)10.1063/1.4929520 (DOI)000360311900030 ()2-s2.0-84940909670 (Scopus ID)
Forskningsfinansiär
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Merknad

QC 20150921

QC 20191008

Tilgjengelig fra: 2015-09-21 Laget: 2015-09-18 Sist oppdatert: 2019-10-08bibliografisk kontrollert
2. RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae
Åpne denne publikasjonen i ny fane eller vindu >>RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae
Vise andre…
2019 (engelsk)Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 116, nr 19, s. 9324-9332Artikkel i tidsskrift (Fagfellevurdert) 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.

sted, utgiver, år, opplag, sider
National Academy of Sciences, 2019
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-250438 (URN)10.1073/pnas.1820561116 (DOI)000467226400031 ()2-s2.0-85065621578 (Scopus ID)
Merknad

QC 20190529

Tilgjengelig fra: 2019-04-29 Laget: 2019-04-29 Sist oppdatert: 2019-09-30
3. Droplet microfluidic screening of a Synechocystis sp. CRISPRi library based on L-lactate production
Åpne denne publikasjonen i ny fane eller vindu >>Droplet microfluidic screening of a Synechocystis sp. CRISPRi library based on L-lactate production
Vise andre…
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-259487 (URN)
Merknad

QC 20191011

Tilgjengelig fra: 2019-09-16 Laget: 2019-09-16 Sist oppdatert: 2019-10-11bibliografisk kontrollert
4. Droplet microfluidic microcolony analysis of triacylglycerol yields in S. cerevisiae for high throughput screening
Åpne denne publikasjonen i ny fane eller vindu >>Droplet microfluidic microcolony analysis of triacylglycerol yields in S. cerevisiae for high throughput screening
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-259488 (URN)
Tilgjengelig fra: 2019-09-16 Laget: 2019-09-16 Sist oppdatert: 2019-10-18bibliografisk kontrollert

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