Cristin-resultat-ID: 1618157
Sist endret: 12. august 2019, 20:13
Resultat
Poster
2018

A new versatile shuttle vector for genome engineering of Bacillus subtilis by the CRISPR/Cas9 system

Bidragsytere:
  • Antonio Garcia-Moyano
  • Øivind Larsen og
  • Gro Elin Kjæreng Bjerga

Presentasjon

Navn på arrangementet: Digital Life Annual Conference
Sted: Bergen
Dato fra: 20. mars 2018
Dato til: 21. mars 2018

Arrangør:

Arrangørnavn: Centre for Digital Life Norway

Om resultatet

Poster
Publiseringsår: 2018

Beskrivelse Beskrivelse

Tittel

A new versatile shuttle vector for genome engineering of Bacillus subtilis by the CRISPR/Cas9 system

Sammendrag

Introduction: The well-known gram-positive bacterium Bacillus subtilis is an attractive host for the production of heterologous proteins and it has therefore been object of some of the recent developments in bacterial genome engineering by CRISPR/Cas9 technology (Zhang et al., 2016; Altenbuchner, 2017). The different editing systems recently developed are based on a single broad host-range plasmid for creating mutant strains with improved fermentation capabilities. Due to the need of specific target sequences and repair templates for homologous recombination, obtaining the final knockout plasmid often requires several cloning steps. Methods: By means of restriction-free cloning techniques, we have adapted an existing shuttle plasmid by introducing a counter-selection cassette compatible with fragment exchange (FX) cloning technology (Geertsma and Dutzler, 2011). In order to test this technology we targeted the spoIIIAC gene that encodes a sigma factor which has an essential role in controlling sporulation. A editing cassette was designed, synthesized and efficiently cloned into the new pCC9 vector by FX-cloning. The verified plasmid was then used for transformation in the protease-deficient B. subtilis K07. Positive transformant clones were screened and correct mutation confirmed. Results and Discussion: The method proved to be very efficient as 19 of 20 screened clones were correctly engineered. Further sequencing confirmed the disruption of spoIIIAC. The new knockout strain K07S2 lacks the ability to form spores when heat-treated in minimal medium (see figure). Our new versatile vector pCC9 allows rapid and effective generation of a knockout plasmid for transformation and genome engineering in these industrially relevant strains.

Bidragsytere

Antonio Garcia-Moyano

  • Tilknyttet:
    Forfatter
    ved NORCE Norwegian Research Centre AS

Øivind Larsen

  • Tilknyttet:
    Forfatter
    ved NORCE Klima og miljø ved NORCE Norwegian Research Centre AS
Aktiv cristin-person

Gro Elin Kjæreng Bjerga

  • Tilknyttet:
    Forfatter
    ved NORCE Klima og miljø ved NORCE Norwegian Research Centre AS
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