MOESM1 of Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum
Creators: name: Liu, Jiao
name: Wang, Yu
name: Yujiao Lu
name: Zheng, Ping
name: Jibin Sun
name: Yanhe Ma
Publisher: name: Figshare
Year: 2017
Subjects: subject: Biochemistry
subject: Space Science
subject: Microbiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Genetics
subject: Molecular Biology
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: Ecology
subject: Developmental Biology
subject: Marine Biology
subject: Cancer
subject: Hematology
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences

description: Additional file 1: Figure S1. PCR verification of ldhA deletion using CRISPR/Cas9 and the two-step electrotransformation strategy in Corynebacterium glutamicum SL4. M, DNA marker; –, wild-type control; 1–20, twenty colonies. pCas9 was firstly transformed into C. glutamicum SL4. pgRNA3 was then transformed into strain SL4 (pCas9) and cells were spread on SGY plates supplemented with Km, Cm and IPTG (1 mM) immediately after recovery. After cultivated at 30 °C, twenty small colonies instead of the abnormally large colonies were picked to perform colony PCR using a pair of primers ldhA-up and ldhA-down. Figure S2. PCR verification of ldhA deletion using CRISPR/Cas9 and the one-step electrotransformation strategy in C. glutamicum SL4. (a) Replicate 2. M, DNA marker; –, wild-type control; 1–6, six colonies. (b) Replicate 3. M, DNA marker; –, wild-type control; 1–9, nine colonies. pCas9 and pgRNA3 were co-transformed into C. glutamicum SL4 simultaneously and cells were spread on SGY plates supplemented with Km, Cm and IPTG (1 mM) immediately after recovery. After cultivated at 30 °C, all the small colonies on the plates were picked to perform colony PCR using a pair of primers ldhA-up and ldhA-down. Figure S3. PCR verification of ldhA deletion using pCas9 and pgRNA3-derivative plasmid that harbored no targeting spacer in C. glutamicum SL4. M, DNA marker; –, wild-type control; 1–20, twenty colonies. pCas9 and pgRNA3-derivative plasmid that harbored no targeting spacer were co-transformed into C. glutamicum SL4 simultaneously and cells were spread on SGY plates supplemented with Km, Cm and IPTG (1 mM) immediately after recovery. After cultivated at 30 °C, twenty colonies on the plates were picked to perform colony PCR using a pair of primers ldhA-up and ldhA-down. Figure S4. PCR verification of rfp insertion using CRISPR/Cas9 and plasmid-borne editing template in C. glutamicum SL4. (a) Replicate 2. M, DNA marker; –, wild-type control; 1–13, thirteen colonies. (b) Replicate 3. M, DNA marker; –, wild-type control; 1–9, nine colonies. pCas9 and pgRNA4 were co-transformed into C. glutamicum SL4 simultaneously and cells were spread on SGY plates supplemented with Km, Cm and IPTG (1 mM) immediately after recovery. After cultivated at 30 °C, all the small colonies on the plates were picked to perform colony PCR using a pair of primers rfp-up and ldhA-down. Figure S5. Transformants harboring pCas9, pgRNA5, and ssDNA (rfp-off1). Plasmid pgRNA5 was first transformed into C. glutamicum SL4ΔldhA::rfp. The resultant strain SL4ΔldhA::rfp (pgRNA5) was cultivated with addition of propionate to induce RecT expression, and then electrocompetent cells were prepared. Next, cells were transformed with pCas9 and ssDNA (rfp-off1) and spread on SGY plates supplemented with Km, Cm and IPTG (1 mM) immediately after recovery. Figure S6. Fluorescence output detection of candidate mutants of C. glutamicum SL4ΔldhA::rfp off1. (a) Replicate 2. –, wild-type C. glutamicum SL4 control; +, C. glutamicum SL4ΔldhA::rfp; 1–10, ten colonies. (b) Replicate 3. –, wild-type C. glutamicum SL4 control; +, C. glutamicum SL4ΔldhA::rfp; 1–10, ten colonies. Figure S7. Fluorescence output detection of candidate mutants of C. glutamicum ATCC 13032::rfp off1. –, wild-type C. glutamicum ATCC 13032 control; +, C. glutamicum ATCC 13032::rfp; 1–10, ten colonies. Figure S8. Fluorescence output detection of candidate mutants of C. glutamicum ATCC 13032::rfp off2. –, wild-type C. glutamicum ATCC 13032 control; +, C. glutamicum ATCC 13032::rfp; 1–10, ten colonies. Table S1. Sequences of primers and ssDNAs used in this study.