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Table 2 Strains and plasmids used in this study

From: Development and characterization of a CRISPR/Cas9n-based multiplex genome editing system for Bacillus subtilis

Name

Relevant genotype

Source/reference

Strains

 B. subtilis 168

Wild-type strain, trpC2

BGSCa

 B. subtilis 168ΔligD

B. subtilis 168ΔligD

This study

 BS89

B. subtilis168Δupp ribC*ribO*

yhcF*yvrH*ywaA* P43::ribA

[55]

 BS89ΔligD

BS89ΔligD

This study

 CY series strains

Modulation library strains generated with CRISPR/Cas9n technique

This study

 E. coli. DH5α

F−, φ80lacZΔM15, Δ(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk−, mk+), phoA, supE44, λ−, thi-1, gyrA96, relA1

Lab stock

Plasmids

 pCas9cur

E. coli cloning vector for the express Cas9 protein, AmpR

Lab stock

 pBSCas9

B. subtilis/E. coli shuttle vector, CmR

PxylA-Cas9, P43-gRNA

This study

 pBSCas9n

pBSCas9 harboring Cas9 mutation

This study

 pDonor

B. subtilis/E. coli shuttle vector, CmR

AmpR, Pmanp-rep60gRNA

This study

 pHG

Derived from pUC18 for multiple gRNA construction

This study

 pUC18

E. coli cloning vector, AmpR

Lab stock

 pEBs-cop1

B. subtilis/E. coli shuttle vector, EmR, AmpR

Lab stock

 pAX01

B. subtilis integration vector, PxylA, EmR, AmpR

Lab stock

 pHP13

B. subtilis/E.coli shuttle vector,CmR, EmR

Lab stock

 pBSCas9-amyEgRNA

Derived from pBSCas9 for targeting amyE

This study

 pBSCas9n-amyEgRNA

Derived from pBSCas9n for targeting amyE

This study

 pBSCas9-Ins1 kbamyEgRNA

Derived from pBSCas9 for targeting Δ1kbamyE

This study

 pBSCas9n-Ins1kbamyEgRNA

Derived from pBSCas9n for targeting Δ1kbamyE

This study

 pBSCas9-Ins2kbamyEgRNA

Derived from pBSCas9 for targeting Δ2kbamyE

This study

 pBSCas9n-Ins2kbamyEgRNA

Derived from pBSCas9n for targeting Δ2kbamyE

This study

 pBSCas9-amyE/uppgRNA

Derived from pBSCas9 for targeting amyE and upp

This study

 pBSCas9n-amyE/uppgRNA

Derived from pBSCas9n for targeting amyE and upp

This study

 pBSCas9-amyE/upp/sigEgRNA

Derived from pBSCas9 for targeting amyE,upp and sigE

This study

 pBSCas9n-amyE/upp/sigEgRNA

Derived from pBSCas9n for targeting amyE,upp and sigE

This study

 pBSCas9-LGgRNA

Derived from pBSCas9 for targeting large DNA fragment

This study

 pBSCas9n-LGgRNA

Derived from pBSCas9n for targeting large DNA fragment

This study

 pDonor-Del1kb

Derived from pDonor for 1 kb deletion

This study

 pDonor-Del2kb

Derived from pDonor for 2 kb deletion

This study

 pDonor-Del4kb

Derived from pDonor for 4 kb deletion

This study

 pDonor-Del6kb

Derived from pDonor for 6 kb deletion

This study

 pDonor-Del8kb

Derived from pDonor for 8 kb deletion

This study

 pDonor-Ine1kb

Derived from pDonor for 1 kb insertion

This study

 pDonor-Ine2kb

Derived from pDonor for 2 kb insertion

This study

 pDonor-DelLDNA

Derived from pDonor for large DNA fragment deletion

This study

 pDonor-MuamyE

Derived from pDonor for codon replacement in amyE

This study

 pDonor-MuamyE/upp

Derived from pDonor for codon replacement in amyE and upp

This study

 pDonor-MuamyE/upp/sigE

Derived from pDonor for codon replacement in amyE,upp and sigE

This study

 pBSCas9n-gRNArib

Derived from pBSCas9n for targeting ribB, ribA and ribH

This study

 pDonor-ribRBSLib

Derived from pDonor for generating a combinatorial library

This study

  1. Cm chloramphenicol; Amp ampicillin; Em erythromycin; R resistance
  2. aBacillus Genetic Stock Center