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Metabolite profiling of microfluidic cell culture conditions for droplet based screening
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.ORCID-id: 0000-0002-3722-5970
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.ORCID-id: 0000-0001-5232-0805
2015 (Engelska)Ingår i: Biomicrofluidics, ISSN 1932-1058, E-ISSN 1932-1058, Vol. 9, nr 4, artikel-id 044128Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
2015. Vol. 9, nr 4, artikel-id 044128
Nyckelord [en]
Biomolecules;Cell culture;Cell proliferation;Cells;Dimensional stability;Glucose;Metabolism;Metabolites;Microfluidics;Yeast
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:kth:diva-173782DOI: 10.1063/1.4929520ISI: 000360311900030Scopus ID: 2-s2.0-84940909670OAI: oai:DiVA.org:kth-173782DiVA, id: diva2:855261
Forskningsfinansiär
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Anmärkning

QC 20150921

QC 20191008

Tillgänglig från: 2015-09-21 Skapad: 2015-09-18 Senast uppdaterad: 2019-10-08Bibliografiskt granskad
Ingår i avhandling
1. Droplet microfluidics for screening and sorting of microbial cell factories
Öppna denna publikation i ny flik eller fönster >>Droplet microfluidics for screening and sorting of microbial cell factories
2019 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
KTH Royal Institute of Technology, 2019. s. 58
Serie
TRITA-CBH-FOU ; 2019:43
Nyckelord
Droplet microfluidics, Cell factories, High-throughput screening, Cell culture
Nationell ämneskategori
Teknik och teknologier
Forskningsämne
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-259490 (URN)978-91-7873-290-6 (ISBN)
Disputation
2019-10-11, Air & Fire, Tomtebodavägen 23A, Solna, 10:00 (Engelska)
Opponent
Handledare
Anmärkning

QC 2019-09-16

Tillgänglig från: 2019-09-16 Skapad: 2019-09-16 Senast uppdaterad: 2019-09-16Bibliografiskt granskad

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Björk, Sara M.Jönsson, Håkan N.

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Björk, Sara M.Sjostrom, Staffan L.Andersson-Svahn, HeleneJönsson, Håkan N.
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Proteomik och nanobioteknologiScience for Life Laboratory, SciLifeLabNanobioteknologi
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Biomicrofluidics
Biokemi och molekylärbiologi

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