Strains
Escherichia coli TOP10 was used as a cloning host. All E. coli carrying pAM1 series plasmids were cultured on LB plates containing ampicillin at 37°C. Bifidobacteria AR668 was cultured anaerobically in BS medium at 37°C. If necessary, erythromycin was added at a concentration of 5μg/ml. All recombinant plasmids enter the competent cells of bifidobacterium by electroporation.
Enzyme and biotechnology toolkit
Phanta Max Super-Fidelity DNA Polymerase and 2× Taq Master Mix (Novozymes Kunming, China) are used for high-fidelity DNA amplification and PCR screening of the required genotypes. The oligonucleotide primers used were synthesized by BGI (Shenzhen, China). Use conventional restriction enzymes and ligases purchased from Takara, Dalian, China to construct plasmids, or use ClonExpress MultiS one-step cloning kit (Vazyme Biotech, Nanjing, China) to assemble plasmids. Plasmid isolation and DNA purification using kits purchased from Axygen (Hangzhou, China).
Plasmid construction
The plasmids and primers used are shown in Table 1 and 2. The CRISPR-Cas9 editing plasmid consists of the following parts: the linearized vector is obtained by double digestion of pAM1-ldh2 with PstⅠand SpeⅠ; the Cas9 gene is obtained by PCR amplification with Cas9-SpeⅠ-F and Cas9-SpeⅠ-R using plcp_0537 as the template; the whole genome of AR668 is used as the template, PCR amplification of 0348-sgRNA-F and 0348-sgRNA-R was used to obtain 20 nt of the target gene 0348; the AR668 whole genome was used as a template, and primers of 0348-up and 0348-down were used for PCR amplification to obtain the homology arms on both sides of the target gene. Note that all 20 nt need to be aligned with the AR668 genome in advance to avoid off-target due to the presence of highly similar sequences.
The sgRNA and artificially synthesized promoter P23 are assembled by overlap PCR to obtain the sgRNA expression cassette. The above fragments are assembled by two rounds of overlap PCR, and the vector and fragments are assembled by a seamless cloning kit. When preparing a knockout plasmid targeting gene 0208, use pLJ2 as a template, and replace the corresponding homology arms on both sides and 20 nt.
Similarly, knockout plasmids with different homology arms are also constructed based on pLJ2, and the homology arms of the corresponding size are replaced with the other originals unchanged. When constructing knockout plasmids with different knockout fragments, only the sequences of the homology arms on both sides of pLJ2 need to be changed.
Construction of the inducible reporter plasmid pLJ9: The linearized vector was obtained by double digestion of pAM1-ldh2 with Nde1 and Spe1. The promoter sequence of β-galactosidase in the AR668 genome was amplified by pAM1- lacZ-F and pAM1- lacZ-R, and amplified by pAM1-gfp-F and pAM1-gfp-R. The eGFP sequence of pAM1-ldh2-eGFP was increased, the above fragments were connected by overlap PCR, and the vector and fragments were connected by the seamless cloning kit to obtain the recombinant plasmid pLJ9.
The inducible plasmid elimination system consists of the corresponding knockout plasmid and the inducible sgRNA_Em expression cassette. The linearized vector is obtained by single-enzyme digestion of pLJ2 with Xho1, and the lacZ sequence of pAM1-lacZ-eGFP is amplified by sgRNA-Em-lacZ-F and sgRNA-Em-lacZ-R, designed to target Em SgRNA_Em is amplified by sgRNA-Em-F and sgRNA-Em-R, and the two ends are connected to form an inducible sgRNA expression cassette by overlap PCR. The vector and fragments are assembled by seamless cloning kit to construct recombinant plasmid pLJ10.
Competent cell preparation and electroporation
The constructed plasmids were delivered into AR668 by electroporation. The competent cells were prepared as follows. One milliliter of overnight cultures in BS broth were diluted into 50 ml of fresh BS broth supplemented 0.5 mol/L sucrose. The inoculated 50 ml BS broth with the additives were anaerobically incubated at 37 °C until the OD600nm reached at 0.3. The cells were chilled on ice for 30 min and harvested by centrifugation at 5000 rpm and 4°C for 10 min. Cells were washed twice with 0.5 mol/L sucrose and 0.1mmol/L ammonium citrate buffer. The final cell pellet was resuspended in 1ml of ice-cold buffer with 10% glycerol. One hundred-microliter aliquot of the competent cells were stored at -80°C.
The competent cells were mixed with 1000 ng of plasmid DNA and kept on ice for 30 min. Then the mixture was transferred to a pre-cooled Gene Pulser cuvette (Bio-Rad, Hercules, CA, USA). The cuvette was pulsed at various field strengths and parallel resistances using the Gene Pulser Xcell Microbial Electroporation System (Bio-Rad). Following the electroporation, 0.9 ml of BS broth (supplemented with 0.4 mol/L sorbitol, 2 mmol/L calcium chloride and 20 mmol/L magnesium chloride) was added to bacteria and incubated at 37 °C for 4 hours under anaerobic conditions. The bacteria were then plated onto BS agar containing 5 μg/ml of Em. The plates were incubated for more than 48 hours under anaerobic conditions.
Screening and identification of edited genes
The mutants were screened by PCR, and the primers on both sides of the upper and lower homology arms were designed as verification primers. The PCR-correct strains were further identified by sequencing.
Evaluation of homology arms and knockout fragments in the crispr system
In this study, plasmid pLJ2 was used as an example. On this basis, knockout plasmids with upper and lower homology arms of 1000 bp, 500 bp, 250 bp, and 150 bp were constructed, electrotransformed into AR668, and incubated at 37°C for more than 48 hours. The knockout result of the plasmid was verified, and the result was further confirmed by sequencing.
In this study, the plasmid pLJ2 was taken as an example. On this basis, knockout plasmids with knockout fragments of 1000 bp, 300 bp, and 5000 bp were constructed, electrotransformed into AR668, and incubated at 37°C for more than 48 hours. In addition to the results for verification, and further confirm the results by sequencing.
Establishment of eGFP-based inducible reporting system
A fluorescent reporter plasmid containing eGFP was constructed. Based on plasmid pAM1-ldh2, a vector was prepared with Hind Ⅲ and Nde Ⅰ as double restriction sites, lacZ in the AR668 genome was used as a promoter, and eGFP was used as a fluorescent reporter gene to construct a plasmid. The plasmid was transformed into AR668 by electroporation, and a single colony was picked for PCR verification. The single colony containing the correct plasmid was grown in fresh BS broth for lactose induction. The fluorescence is detected by a fluorescence microscope and the relative fluorescence intensity is measured with a microplate reader. The best induction conditions were obtained by optimizing the OD600nm value before induction, the concentration of lactose and the induction time.
Mutant purification and plasmid elimination for continuous genome engineering
Cultivate the mutant strains verified by PCR and sequencing in a resistant medium. For double-banded strains, that is, when the wild type and the mutant type are present at the same time, they should be streaked and separated in the solid medium first, and then the single-banded strains will be verified by colony PCR. The knock-out strains were cultured in non-antibiotic medium. When it is raised to a proper OD600nm, a certain amount of lactose is added to induce. Under the guidance of the sgRNA targeting the erythromycin resistance gene in the plasmid, Cas9 cuts the erythromycin gene to complete the elimination of the plasmid.