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Droplet microfluidic screening of a Synechocystis sp. CRISPRi library based on L-lactate production
KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-3722-5970
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
KTH, Centres, Science for Life Laboratory, SciLifeLab.
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(English)Manuscript (preprint) (Other academic)
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:kth:diva-259487OAI: oai:DiVA.org:kth-259487DiVA, id: diva2:1351544
Note

QC 20191011

Available from: 2019-09-16 Created: 2019-09-16 Last updated: 2019-10-11Bibliographically approved
In thesis
1. Droplet microfluidics for screening and sorting of microbial cell factories
Open this publication in new window or tab >>Droplet microfluidics for screening and sorting of microbial cell factories
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2019. p. 58
Series
TRITA-CBH-FOU ; 2019:43
Keywords
Droplet microfluidics, Cell factories, High-throughput screening, Cell culture
National Category
Engineering and Technology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-259490 (URN)978-91-7873-290-6 (ISBN)
Public defence
2019-10-11, Air & Fire, Tomtebodavägen 23A, Solna, 10:00 (English)
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Note

QC 2019-09-16

Available from: 2019-09-16 Created: 2019-09-16 Last updated: 2019-09-16Bibliographically approved

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Björk, SaraShabestary, KiyanYao, LunJönsson, HåkanHudson, Elton P.

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CiteExportLink to record
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