Ändra sökning
RefereraExporteraLänk till posten
Permanent länk

Direktlänk
Referera
Referensformat
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annat format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annat språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf
A universal platform for selection and high-resolution phenotypic screening of bacterial mutants using the nanowell slide
Karolinska Inst, Dept Neurosci, Swedish Med Nanosci Ctr, Stockholm, Sweden..
Karolinska Inst, Dept Neurosci, Swedish Med Nanosci Ctr, Stockholm, Sweden..
KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
Visa övriga samt affilieringar
2018 (Engelska)Ingår i: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 18, nr 12, s. 1767-1777Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The Petri dish and microtiter plate are the golden standard for selection and screening of bacteria in microbiological research. To improve on the limited resolution and throughput of these methods, we developed a universal, user-friendly platform for selection and high-resolution phenotypic screening based on the nanowell slide. This miniaturized platform has an optimal ratio between throughput and assay complexity, holding 672 nanowells of 500 nl each. As monoclonality is essential in bacterial genetics, we used FACS to inoculate each nanowell with a single bacterium in 15 min. We further extended the protocol to select and sort only bacteria of interest from a mixed culture. We demonstrated this by isolating single transposon mutants generated by a custom-made transposon with dual selection for GFP fluorescence and kanamycin resistance. Optical compatibility of the nanowell slide enabled phenotypic screening of sorted mutants by spectrophotometric recording during incubation. By processing the absorbance data with our custom algorithm, a phenotypic screen for growth-associated mutations was performed. Alternatively, by processing fluorescence data, we detected metabolism-associated mutations, exemplified by a screen for -galactosidase activity. Besides spectrophotometry, optical compatibility enabled us to perform microscopic analysis directly in the nanowells to screen for mutants with altered morphologies. Despite the miniaturized format, easy transition from nano- to macroscale cultures allowed retrieval of bacterial mutants for downstream genetic analysis, demonstrated here by a cloning-free single-primer PCR protocol. Taken together, our FACS-linked nanowell slide replaces manual selection of mutants on agar plates, and enables combined selection and phenotypic screening in a one-step process. The versatility of the nanowell slide, and the modular workflow built on mainstream technologies, makes our universal platform widely applicable in microbiological research.

Ort, förlag, år, upplaga, sidor
ROYAL SOC CHEMISTRY , 2018. Vol. 18, nr 12, s. 1767-1777
Nationell ämneskategori
Mikrobiologi
Identifikatorer
URN: urn:nbn:se:kth:diva-231698DOI: 10.1039/c8lc00190aISI: 000435115300009PubMedID: 29781496Scopus ID: 2-s2.0-85048406946OAI: oai:DiVA.org:kth-231698DiVA, id: diva2:1241747
Anmärkning

QC 20180824

Tillgänglig från: 2018-08-24 Skapad: 2018-08-24 Senast uppdaterad: 2018-08-24Bibliografiskt granskad

Open Access i DiVA

Fulltext saknas i DiVA

Övriga länkar

Förlagets fulltextPubMedScopus

Personposter BETA

Svahn Andersson, Helene

Sök vidare i DiVA

Av författaren/redaktören
Weibull, EmelieSvahn Andersson, Helene
Av organisationen
ProteinvetenskapNanobioteknologi
I samma tidskrift
Lab on a Chip
Mikrobiologi

Sök vidare utanför DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetricpoäng

doi
pubmed
urn-nbn
Totalt: 404 träffar
RefereraExporteraLänk till posten
Permanent länk

Direktlänk
Referera
Referensformat
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annat format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annat språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf