E. coli Strains
DH5α (TransGen, Beijing) was used for cloning plasmid. BL21(DE3) (TransGen, Beijing) was used for cloning T7 RNA polymerase genes, LacI and LacO (T7-RNAP), from its chromosomal genome. BW25113 strains were used for CRISPR/Cas9-induced DSB and recombination. Without especially explanations, all E. coli strains were routinely cultured in standard LB medium.
Selection Of Integration Site And Design Of Homologous Recombination
Sequence of T7-RNAP in BL21(DE3) genome and the integration site of BW25113 were both confirmed in NCBI. The function and detailed message of the ybhC gene was verified in NCBI and BioCyc Database. The N20 site was found by BROAD international design tool, which is available at: http://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design.
Plasmids Construction for CRISPR and preparation of linear Donor dsDNA by PCR
Table 1 contains all primer pairs we designed for gene cloning and intermediate plasmid construction. Plasmid pCas (Fig. 3A) and pTarget were prepared in our laboratory. Plasmid pTarget-ybhC was constructed by Inverse-PCR and T4-Ligation (T4 DNA Ligase, NEB, England) to replace the N20 fragment (Fig. 3B) with specific primers (Table 1). Plasmid pACYCD-ybhC and pACYCD-Donor were both constructed for preparing Donor DNA (Fig. 4) by In-Fusion® HD Cloning Kit (Takara, Japan). Donor DNA was cloned from pACYCD-Donor by Hi-Fi PCR (Phusion® High-Fidelity PCR Master Mix, NEB, England).
All plasmids used in this research will be listed in Table 2.
Electroporation, Cell Recovery, And Plating
For transformation, the plasmid or linear DNA were electroporated into competent cells in the pre-chilled cuvette (0.1 cm) using Bio-Rad MicroPulser (1.8 kV, time constant > 5.0 ms). For selection, 25 µg/mL chloramphenicol (Chl) or 50 µg/mL kanamycin (Kan) were used alone or in combination. For induction of λ-Red proteins and lac operator, 1 mM arabinose and 1 mM IPTG were used.
To prepare cells harboring pCas, cells cultured at 37℃ (OD600 = 0.45–0.55) were made competent, mixed with pCas (100 ng) and subjected to electroporation, after which the cells were recovered in SOC medium (1 mL) for 1 h at 30℃, plated onto the Kan plate, and cultured at 30 ℃ for 18–24 h.
For CRISPR/Cas9-mediated homologous recombination, cells harboring pCas were cultured at 30℃ in medium containing Kan and Arabinose and made competent. After co-electroporation of Donor DNA (400 ng) with pTarget-ybhC (100 ng), cells were recovered in SOC (1 mL) medium for 1 h at 30℃, plated onto Chl/Kan plate, and cultured at 30℃ for 18–24 h.
For elimination of pTarget-ybhC, cells harboring both pCas and pTarget were cultured at 30℃ in medium containing Kan and IPTG for 2 h. Cells were plated onto Kan plates and cultured at 30℃ for 18–24 h.
For elimination of pCas, cells harboring pCas were cultured at 37℃ in the medium without any antibiotic for 12–16 h. Then the cells were plated onto non-antibiotic plates and cultured at 37℃ for 12–16 h.
Verification Of Gene Integration By Colony PCR And DNA-SEQ
To verify correct integration of T7-RNAP, we designed Primer Seq-F and Primer Seq-R as shown in Table S1. Primer Seq-F targeted downstream of the ybhC gene (1267–1288 bp), while Primer Seq-R targeted upstream of the ybhC gene (261–282 bp). If integration was successful, colony PCR of the recombinants would yield a ≈ 5 kb amplicon, which contains HRL, T7-RNAP and HRR. Absences or mutations of the integration fragment were verified by DNA-sequencing.
All DNA-sequencing for this research was conducted by Beijing Ruibio BioTech Co., Ltd (China, Beijing).
Growth Of Bacteria In Different Medium
All E. coli strains were grown at 37℃ in conical flask (250 mL) containing M9 medium (100 mL), M9YE medium or LB medium with chloramphenicol (25 µg/mL). Growth was measured by monitoring optical density at 600 nm (OD600) using a spectrophotometer.
Confirmation of T7 Expression System in BW25113-T7 by sYFP Reporter Gene System
Efficiency of the T7 expression system was confirmed by a fluorescent protein reporter assay system. The plasmid was named as pACYCD-sYFP, which harbored ori-p15a, Chloramphenicol resistance (Chlr), LacI gene, T7-Lac promoter [27] and sYFP gene (Fig. 4). This fluorescent protein could reflect 540 nm light by the 503 nm exciting light. This optical signal could be observed microscopically by Confocal Microscopy and accurately detected by a Fluorescence Detector. This plasmid was transfected into E. coli BL21(DE3) (as Positive Control), E. coli BW25113 (as Negative Control) and E. coli BW25113-T7.
Confocal Microscopy
Cells harboring pACYCD-sYFP were cultured at 37℃ for 2 hours. Then IPTG with a final concentration of 1 mM was added to the induced group. The bacterium solution was observed under Confocal Microscopy after 4 hours of induction.
All strains were prepared for Confocal Microscopy as stated above for fluorescence measurements and prepared as a wet mount. Confocal microscopy was performed using a Leica TCS SP2 inverted confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with a 63× water-corrected objective in multitrack mode. Collected images were analyzed with the Leica Application Suite for Advanced Fluorescence (LAS AF, version 2.35) software (Leica Microsystem).
Fluorescence Intensity Measured By Multifunctional Microplate Detector
Cells harboring pACYCD-sYFP were cultured in LB containing Chl at 37℃ for 2 hours. Then IPTG with a final concentration of 1 mM was added to the induced group. The fermented liquid was added to a 96 well plate after 4 hours of induction, then accurate fluorescent data was detected by Synergy™ HTX Multifunctional Microplate Detector (BioTek Instruments, America). The excitation light was set as 503 nm and the receiving light was set as 540 nm. Collected data was analyzed with the Gen5™ V2 Data Analysis Software (BioTek Instruments, Inc.).
Analytical Procedures Of ALA Production
Cells harboring pET28b-ALA-LA or pET28b-ALA-LAR were cultured in M9YE containing Kan at 37℃ for 2 hours. Then IPTG with a final concentration of 0.1 mM was added to the induced group. To analyze ALA production, culture (30 mL) after induction for 24 h was centrifuged (12,000 g for 2 min at 4℃). The supernatant was used for extracellular ALA analysis. ALA concentration was analyzed using modified Ehrlich’s reagent [48].