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Table 2 Strains and plasmids used in this study

From: Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum

Strain or plasmid

Descriptiona

Reference or source

E. coli

 DH5α

General cloning host

TaKaRa

 MG1655

Source of recT

Lab collection

C. glutamicum

 ATCC 13869

Wild-type strain

ATCC

 ATCC 13032

Wild-type strain

ATCC

 ATCC 13032::rfp

ATCC 13032 derivative with insertion of a rfp cassette

Lab collection

 SL4

ATCC 13869 derivative with high electrotransformation efficiency

Lab collection

 SL4 (pCas9)

SL4 derivative harboring pCas9

This work

 SL4ΔldhA

SL4 derivative with ldhA deletion

This work

 SL4ΔldhA::rfp

SL4 derivative with insertion of a rfp cassette in ldhA

This work

 SL4ΔldhA::rfp (pgRNA5)

SL4ΔldhA::rfp derivative harboring pgRNA5

This work

 SL4ΔldhA::rfp off1

SL4ΔldhA::rfp derivative with nonsense mutation in rfp, constructed by ssDNA (rfp-off1) recombineering

This work

 SL4 (pgRNA6)

SL4 derivative harboring pgRNA6

This work

 SL4 rpsL K43R

SL4 derivative with K43R mutation of rpsL

This work

 SL4 (pcas9 + pgRNA1)

SL4 derivative harboring pcas9 and pgRNA1

This work

 SL4 (pcas9 + pgRNA2)

SL4 derivative harboring pcas9 and pgRNA2

This work

 ATCC 13869ΔldhA

ATCC 13869 derivative with ldhA deletion

This work

 ATCC 13032ΔldhA

ATCC 13032 derivative with ldhA deletion

This work

 ATCC 13032Δcgl1776-cgl178

ATCC 13032 derivative with cgl1776-cgl178 deletion

This work

 ATCC 13032::lacZ

ATCC 13032 derivative with lacZ insertion

This work

 ATCC 13032::rfp (pgRNA5)

ATCC 13032::rfp derivative harboring pgRNA5

This work

 ATCC 13032::rfp off1

ATCC 13032::rfp derivative with nonsense mutation in rfp, constructed by ssDNA (rfp-off1) recombineering

This work

 ATCC 13032::rfp off2

ATCC 13032::rfp derivative with nonsense mutation in rfp, constructed by ssDNA (rfp-off2) recombineering

This work

Plasmid

 pEC-XK99E

Expression vector of C. glutamicum, IPTG-inducible promoter P trc , KmR

[55]

 pXMJ19

Expression vector of C. glutamicum, IPTG-inducible promoter P tac , CmR

[56]

 pRed_Cas9_recA_Δpoxb300

Source of cas9 gene and gRNA fragment

[54]

 pEC-XK99E-rfp

Source of rfp gene

Lab collection

 pCas9

pXMJ19 derivative carrying cas9 gene, driven by IPTG-inducible promoter P tac

This work

 pRfp1

pXMJ19 derivative carrying cas9 180bp-rfp artificial gene, driven by IPTG-inducible promoter P tac

This work

 pRfp2

pXMJ19 derivative carrying cas9 180bp-rfp artificial gene, driven by propionate-inducible promoter P prpD2

This work

 pRfp3

pEC-XK99E derivative carrying gRNA-rfp cassette, driven by constitutive promoter P 11F

This work

 pRfp4

pEC-XK99E derivative carrying gRNA (with T sp deleted)-rfp cassette, driven by constitutive promoter P 11F

This work

 pgRNA1

pEC-XK99E derivative carrying gRNA targeting ldhA, driven by IPTG-inducible promoter P tac

This work

 pgRNA2

pEC-XK99E derivative carrying gRNA targeting ldhA, driven by constitutive promoter P 11F , followed by T rrnB without the 185 bp redundant segment

This work

 pgRNA3

pgRNA2 derivative carrying homologous arms (~ 1 kb) for ldhA deletion and corresponding gRNA cassette

This work

 pgRNA4

pgRNA3 derivative with a rfp cassette inserted between the two homologous (~ 1 kb) arms for replacing ldhA with the rfp cassette

This work

 pgRNA5

pgRNA2 derivative carrying gRNA targeting rfp and a P prpD2 -recT cassette for ssDNA-directed rfp editing

This work

 pgRNA6

pgRNA5 derivative carrying gRNA targeting rpsL and a P prpD2 -recT cassette for ssDNA-directed rpsL editing

This work

 pgRNA7

pgRNA5 derivative carrying two gRNAs targeting rfp and rpsL and a P prpD2 -recT cassette for double-locus editing

This work

 pgRNA8

pgRNA3 derivative carrying homologous arms (~ 0.5 kb) for ldhA deletion and corresponding gRNA cassette

This work

 pgRNA9

pgRNA3 derivative carrying homologous arms (~ 1 kb) for cgl1776-cgl1781 deletion (8083 bp) and corresponding gRNA cassette

This work

 pgRNA10

pgRNA3 derivative carrying homologous arms (~ 1 kb), lacZ cassette (3626 bp) and corresponding gRNA cassette for lacZ cassette insertion

This work

  1. aKmR and CmR represent resistance to kanamycin and chloramphenicol, respectively