Sep 04, 2025

Public workspaceRecombineering-assisted linear CRISPR/Cas9-mediated multiplex genome editing (ReaL-MGE) for bacterial metabolic engineering

DOI
dx.doi.org/10.17504/protocols.io.3byl4693zgo5/v1
  • Wentao Zheng1,
  • Hao Sun1,
  • Qiang Tu1,
  • Xiaoying Bian1,
  • Youming Zhang1,
  • wangxue 1
  • 1State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong 266237, P.R.China
wangxue
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.3byl4693zgo5/v1
External link: https://doi.org/10.1073/pnas.1720941115; https://doi.org/10.1093/nar/gkab1076; https://doi.org/10.1038/s41467-024-54191-4
Protocol Citation: Wentao Zheng, Hao Sun, Qiang Tu, Xiaoying Bian, Youming Zhang, wangxue 2025. Recombineering-assisted linear CRISPR/Cas9-mediated multiplex genome editing (ReaL-MGE) for bacterial metabolic engineering. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4693zgo5/v1
Manuscript citation:
No
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 26, 2025
Last Modified: September 04, 2025
Protocol Integer ID: 225489
Keywords: recombineering, CRISPR/Cas9, multiplex genome engineering, malonyl-CoA, lignocellulose, alternative bacterial multiplex genome editing technology, multiplex genome editing in gammaproteobacteria, mediated multiplex genome editing, bacterial genome engineering, scope of bacterial genome engineering, multiplex genome editing, significant advancement in bacterial genetic engineering, programmability of crispr, bacterial genetic engineering, assisted linear crispr, bacterial metabolic engineering recombineering, linear crispr, potential for diverse synthetic biology application, scale dna manipulation at multiple genomic loci, phage recombinase, diverse synthetic biology application, crispr, protein activities of phage recombinase, multiple genomic loci, scale dna manipulation, escherichia coli, gammaproteobacteria, mge mitigate, distinct genomic loci, assembling multiple grna, biotechnology, annealing protein activity
Funders Acknowledgements:
the National Key R&D Program of China
Grant ID: 2023YFC3402000
the National Natural Science Foundation of Shandong province
Grant ID: ZR2023YQ028
Taishan Scholar Program of Shandong Province in China
Grant ID: tsqn202312007
Disclaimer
The authors declare no competing interests.
Abstract
Recombineering-assisted Linear CRISPR/Cas9-mediated Multiplex Genome Editing (ReaL-MGE) constitutes a significant advancement in bacterial genetic engineering. This technology synergizes the RNA-guided programmability of CRISPR/Cas9 with the 5’-3’ exonuclease and single-strand DNA annealing protein activities of phage recombinases, enabling precise kilobase-scale DNA manipulation at multiple genomic loci simultaneously. ReaL-MGE mitigates off-target effects, removes substrate restrictions, and circumvents the complexities associated with assembling multiple gRNAs on circular vectors. The development of successive ReaL-MGE iterations addresses bacterial intolerance to simultaneous multi-site genomic editing. ReaL-MGE enables the precise simultaneous integration of 22 kilobase-scale sequences (>1 kb each) into distinct genomic loci of non-model bacteria, thereby expanding the scope of bacterial genome engineering. Demonstrating cross-class applicability, ReaL-MGE facilitates multiplex genome editing in Gammaproteobacteria (Escherichia coli and Pseudomonas putida) and Betaproteobacteria (Schlegelella brevitalea), highlighting its potential for diverse synthetic biology applications in biotechnology, agriculture, and environmental science. Compared to alternative bacterial multiplex genome editing technologies, ReaL-MGE offers significant advantages, including unrestricted editing sites and positions, no substrate length limitation, and enhanced convenience and safety. This study provides a comprehensive protocol detailing ReaL-MGE’s capabilities, demonstrating its superiority over prior multi-site editing techniques and its potential to transform multiplex genome engineering. The entire procedure entails approximately 9 days.
Image Attribution
No
Guidelines
Steps 1-10 (day 2), construction of expression and biosensor plasmids: 3 h
Steps 11-19 (day 2), electroporation of expression and biosensor plasmids: 3 h
Steps 20-22 (day 1), verification of expression plasmid and biosensor transformation: 4 h
Steps 23-29 (day 2), seamless modifications by ReaL-MGE: 12 h
Steps 30-40 (day 1), FACS sorting for GFP expression from the FapR biosensor: 5 h
Steps 41-48 (day 1), malonyl-CoA quantification: 2 h
Materials
Materials
Biological Materials
▲CRITICAL For a summary of all strains used in this protocol.
• Escherichia coli BL21 (DSM102052) is a Gram-negative bacterium classified within the domain Bacteria, phylum Pseudomonadota (synonym: Proteobacteria), class Gammaproteobacteria, order Enterobacterales, family Enterobacteriaceae, genus Escherichia, and species Escherichia coli. This strain is extensively utilized in biotechnological applications due to its robustness, rapid growth kinetics, and high recombinant protein expression capacity. E. coli BL21 possesses Redγβα homologous recombinases that enable high-fidelity genome editing.
• Schlegelella brevitalea (DSM7029) is a Gram-negative bacterium classified within the domain Bacteria, phylum Pseudomonadota (synonym: Proteobacteria), class Betaproteobacteria, order Burkholderiales, family Comamonadaceae, genus Schlegelella, and species Schlegelella brevitalea. In bioindustrial applications, S. brevitalea DSM7029 is notable for its potential in biosynthesis due to its metabolic versatility. S. brevitalea DSM7029 possesses Redβα7029 homologous recombinases that enable high-fidelity genome editing.
• Pseudomonas putida KT2440 (DSM26250) is a Gram-negative bacterium classified within the domain Bacteria, phylum Pseudomonadota, class Gammaproteobacteria, order Pseudomonadales, family Pseudomonadaceae, genus Pseudomonas, and species Pseudomonas putida. Recognized as a preferred chassis organism for synthetic biology due to its genomic stability and genetic tractability, P. putida KT2440 has been extensively engineered for diverse applications including environmental bioremediation, biosynthesis of value-added compounds (e.g., bioplastics, fine chemicals), and plant growth promotion. P. putida KT2440 possesses BAS homologous recombinases that enable high-fidelity genome editing.
• E. coli strain GB05-dir (Gene Bridges) carries Rac recombinases RecET (ETγA) on the genome, which are regulated by arabinose promoters.
Reagents
Plasmids
▲CRITICAL For a summary of all plasmids used in this protocol.
• pBBR1-PRha-Redγβα-xseBecoli-PBAD-Cas9-xseAi-Km.This plasmid with a broad-host-range pBBR1 origin. This construct encodes the arabinose-inducible cas9,rhamnose-inducible redγβα operon (redα, redβ and redγ) and xseBecolifrom E. coli, cassette for xseA inactivation and conveys kanamycin resistance.
• pBBR1-PRha-Redγ-Redαβ7029-xseB7029-xseAi-Km.This plasmid with a broad-host-range pBBR1 origin. This construct encodes the rhamnose-inducible redγ-redαβ7029operon (redα7029, redβ7029 and redγecoli) and xseB7029from S. brevitalea, cassette for xseA inactivation and conveys kanamycin resistance.
• pBBR1-PRha-BAS-xseBkt2440-PBAD-Cas9-xseAi-Km.This plasmid with a broad-host-range pBBR1 origin. This construct encodes the arabinose-inducible cas9,rhamnose-inducible BAS operon (alpha, beta and SSB from phage_AB31), and xseBkt2440 from P. putida, cassette for xseA inactivation and conveys kanamycin resistance.
• RK2-J233-GFP-genta-FapRecoli-amp. This biosensor plasmid is based on a RK2 origin and harbors the green fluorescent protein reporter gene (gfp) and gentamicin resistance gene (genta) under the control of the J233 promoter and codon optimized fapR gene for E. coli.
• RK2-J233-GFP-genta-FapR7029-amp. This biosensor plasmid is based on a RK2 origin and harbors the gfp and genta genes under the control of the J233 promoter and codon optimized fapR gene for S. brevitalea.
• RK2-J233-GFP-genta-FapRkt2440-amp. This biosensor plasmid is based on a RK2 origin and harbors the gfp and genta genes under the control of the J233 promoter and codon optimized fapR gene for P. putida.
• p15A-cm-Cas9-J23119. This plasmid with a p15A origin. This construct encodes the cas9,J23119 promoter, and conveys chloramphenicol resistance.
• QIAquick PCR Purification Kit (Qiagen, cat. no. 28104)
• Qiagen Plasmid Mini Kit (Qiagen, cat. no. 12123)
• Tryptone (Oxoid, cat. no. LP0042)
• Yeast extract (Oxoid, cat. no. LP0021)
• NaCl (Sangon Biotec, cat. no. SB0476)
• NaOH (Sangon Biotec, cat. no. A100583)
• K2HPO4 (Sangon Biotec cat. no. A501212)
• MgSO4 (Sangon Biotec cat. no. A500864)
• DNA 6 × loading buffer (Takara, cat. no. 9156)
• Agar (Solarbio, cat. no. A8190)
• Autoclaved ddH2O kept at room temperature (RT, 18–22 °C) and on ice
• Chloramphenicol (Sigma-Aldrich, cat. no. C1919)
• Kanamycin sulfate (Sigma-Aldrich, cat. no. K4000)
• Gentamicin solution (50 mg mL−1; Sigma-Aldrich, cat. no. G1397)
• Isopropanol (Sinopharm, cat. no. 80109218)
• Ethanol, absolute (Sinopharm, cat. no. 10009218)
• 70% (vol/vol) ethanol (Qiagen, cat. no. AM1091)
• RNase A (10 mg mL−1; DNase and protease free; Thermo Scientific, cat. no. EN0531)
• L-Arabinose (Sigma-Aldrich, cat. no. A3256)
• L-Rhamnose (Sigma-Aldrich, cat. no. 83650)
• Sucrose (Sangon Biotec, cat. no. A502792)
• Glycerol (Sangon Biotec, cat. no. A100854)
• 1-kb DNA ladder (New England BioLabs, cat. no. N3232)
• PrimeSTAR Max DNA Polymerase (Takara, cat. no. R045B)
• 10 × Tris/boric acid/EDTA (TBE) buffer (Bio-Rad, cat. no. 161-070)
• Ethidium bromide solution (10 mg mL-1; Dingguo, cat. no. NEP028-1)
• Agarose (Takara, cat. no. 5261)
• Buffer P1 (Qiagen, cat. no. 19051)
• Buffer P2 (Qiagen, cat. no. 19052)
• Buffer P3 (Qiagen, cat. no. 19053)
• Primers and oligonucleotides (Sangon Biotec): oligomers required for constructing the mutation and detecting the mutation in the Pseudomonas chromosome (see Table 1 for examples)
Equipment
• Thermomixer (Eppendorf, model F1.5, cat. no. 5384000.071)
• MixMate (Eppendorf, cat. no. 022674226)
• Benchtop centrifuge, kept at RT (Eppendorf, model 5424R, cat. no. 5424000.010)
• Benchtop centrifuge, kept at 4°C (Eppendorf, model 5424R, cat. no. 5404000.014)
• Vortex (Scientific Industries, cat. no. G560E)
• Electroporator (Eppendorf, model 2510, cat. no. 940000009)
• Electroporation cuvettes with 1-mm gap, kept on ice (Eppendorf, cat. no. 940001005)
• Petri dishes, 94 mm × 16 mm (Greiner Bio-One, cat. no. 633180)
• Digital gel imaging system (GelDoc XR+, Bio-Rad)
• UV spectrophotometer (NanoDrop 2000c, Thermo Scientific)
• UV-visible spectrophotometer (T6 New Century, Purkinje General Instrument)
• Gel electrophoresis apparatus (Beijing Junyi, cat. no. JY300C)
• Flow cell sorter (BD, cat. no. FACSAria‱ Fusion)
• Multifunctional microplate reader (Agilent, cat. no. BioTek Synergy H1)
• Incubators kept at 30°C and 37°C (Ningbo Jiangnan, cat. no. HWS-0288)
• pH detector (Sartorius, cat. no. PB-10)
• Sterile 10-μL inoculation loops (Sangon Biotec, cat. no. IL311-10-S-Q)
• Sterile 1-μL inoculation loops (Sangon Biotec, cat. no. IL311-1-S)
• 0.22-μm syringe filters (Pall, cat. no. PN4612)
• Millipore membrane filters (Merck-Millipore, cat. no. VSWP01300)
• 1-mL cuvettes (Fisher Scientific, cat. no. 14955127)
• PCR tube strips, 200 μL (Sangon Biotec, cat. no. F601550-0001)
• 1.5-mL microcentrifuge tubes (Sangon Biotec, cat. no. F600620-9001)
• 2.0-mL microcentrifuge tubes (Sangon Biotec, cat. no. F600619-9001)
• 50-mL centrifuge tubes (sterile, DNase/RNase-Free; Sangon Biotec, cat. no. CT788-GS)
• Syringe needles, 25-gauge 5/8, 0.5 mm × 16 mm (BD Medical, cat. no. 301805)
• Multipette (Eppendorf, cat. no. 4981000.019)
Reagent setup
LB broth
Prepare the solution by dissolving 10 g tryptone, 5 g yeast extract, and 1 g NaCl in ~900 mL ultrapure water. Following pH adjustment to 8.0 with 10% NaOH, adjust the volume to 1 L. Sterilize the medium via autoclaving (121°C, 20 min). After allowing it to cool, add antibiotics prior to use.
▲CRITICAL The broth can be kept at room temperature (RT) for several months.
LB agar plates
Prepare the solution by dissolving 10 g tryptone, 5 g yeast extract, 1 g NaCl and 12 g agar in ~900 mL ultrapure water. Following pH adjustment to 8.0 with 10% NaOH, adjust the volume to 1 L. Sterilize the medium via autoclaving (121°C, 20 min). After allowing it to cool, add antibiotics prior to use. Pour 20~25 mL medium into Petri dishes and allow the agar to solidify in a sterile hood.
▲CRITICAL To prevent photodegradation of tetracycline, agar plates supplemented with this antibiotic should be shielded from light during storage, typically by wrapping in aluminum foil.
▲CRITICAL Agar plates are typically prepared aseptically for experimental use. Any unused plates can be stored at 4°C for up to one week.
CYMG broth
Prepare the solution by dissolving 8 g tryptone, 4 g yeast extract, 4.06 g MgCl2·2H2O and 5 mL glycerol in ~900 mL ultrapure water. Following pH adjustment to 8.0 with 10% NaOH, adjust the volume to 1 L. Sterilize the medium via autoclaving (121°C, 20 min). After allowing it to cool, add antibiotics prior to use.
▲CRITICAL The broth can be kept at RT for several months.
CYMG agar plates
Prepare the solution by dissolving 8 g tryptone, 4 g yeast extract, 4.06 g MgCl2·2H2O, 5 mL glycerol and 12 g agar in ~900 mL ultrapure water. Following pH adjustment to 8.0 with 10% NaOH, adjust the volume to 1 L. Sterilize the medium via autoclaving (121°C, 20 min). After allowing it to cool, add antibiotics prior to use. Pour 20~25 mL medium into Petri dishes and allow the agar to solidify in a sterile hood.
0.8% (wt/vol) agarose gels
Dissolve 0.56 g of agarose powder in 70 mL of 1× TBE buffer. Heat the mixture in a microwave oven until the agarose is completely dissolved. Allow the molten agarose solution to cool to approximately 60°C. Add 6 μL of ethidium bromide solution (10 mg mL⁻¹) and mix thoroughly to ensure homogeneity. Cast the solution into a gel tray fitted with an appropriate comb and allow polymerization to occur at ambient temperature until solidified.
Antibiotic stock solutions
Prepare 30 mg mL−1 chloramphenicol solution in 100% (vol/vol) ethanol. Divide 1-mL aliquots into 1.5-mL tubes in a sterile hood and store them at -20°C until needed. Prepare stock solution of gentamycin (5 mg mL−1) and kanamycin (30 mg mL−1) in autoclaved ddH2O. Sterilize the solution by filtration in a sterile hood. Divide 1-mL aliquots into 1.5-mL tubes and store them at −20°C until further use.
10% (wt/vol) L-arabinose and 10% (wt/vol) L-rhamnose
Dissolve 5.0 g of sucrose in approximately 40 mL of sterile, deionized, distilled water (ddH2O). Quantitatively transfer the solution to a 50 mL volumetric flask and bring the final volume to 50.0 mL with additional sterile ddH2O. Sterilize the solution by filtration through a 0.22 μm membrane filter under aseptic conditions within a laminar flow cabinet. Aseptically aliquot 1.0 mL volumes of the sterile solution into pre-sterilized 1.5 mL microcentrifuge tubes. Store aliquots at -20°C for long-term preservation until required.
1 × TBE electrophoresis buffer
Dilute 50 × TBE buffer in ddH2O to a 1 × solution and store it at RT until further use.

Troubleshooting
Safety warnings
No


Ethics statement
No
Before start
Experimental design
Overview of the Procedure
The utilization of ReaL-MGE systems can be divided into four parts (a total of 57 Procedure steps):
• Part 1, preparation of plasmids, strains and reagents
• Part 2, establishment of ReaL-MGE
• Part 3, multiplex malonyl-CoA metabolic and genome engineering in E. coli
• Part 4, application to different hosts


Part 1
Preparation of plasmids, strains and reagents
To establish plasmid systems with recombinase and Cas9 controlled by separate inducible promoters and produce linearized guide RNA (gRNA) cassettes, execute these precisely ordered procedures.
Select bacterial strains based on experimental requirements (e.g., E. coli BL21, S. brevitalea DSM7029, and P. putida KT2440). Verify that candidate strains possess authenticated genetic backgrounds, including well-characterized genome sequences and validated compatibility with specific recombination or transformation experiments.
Select broad-host-range vector systems with verified stability and compatible antibiotic markers. For designated strains (E. coli BL21, S. brevitalea DSM7029, and P. putida KT2440), employ strain-adapted recombination plasmids: pBBR1-PRha-Redγβα-Km-amp-ccdB, pBBR1-PRha-Redγ-Redαβ7029-Km-amp-ccdB, and pBBR1-PRha-BAS-Km-amp-ccdB respectively. Perform ScaI digestion followed by 37°C incubation (3 h) to generate linearized vectors containing rhamnose-inducible promoter systems, recombinases, and antibiotic resistance gene, flanked by homology sequences for precise integration of cas9 gene, xseB gene, and xseA knockout cassette.
Perform PCR amplification using plasmid RK2-PBAD-Cas9-genta as a template with high-fidelity polymerases to generate pBAD-regulated Cas9 expression cassettes. Amplify the PCR products using primers with complementary overhangs and regulatory sequences such as terminators to ensure robust expression. Validate amplification fidelity through 1.2% agarose gel electrophoresis and sequencing.
Amplify xseB gene using strain-specific primers: xseB-ecoli-1/2 for E. coli., xseB-7029-1/2 for S. brevitalea DSM7029, and xseB-kt2440-1/2 for P. putida KT2440. Confirm amplicon sizes using 1.2% agarose gel electrophoresis and validate sequence via sequencing with original PCR primers.
To prepare electrocompetent E. coli GB05-dir (Gene Bridges) for co-transformation with linearized vectors and plasmid elements while inducing RecET recombinase expression, single colonies were streaked onto LB agar plates and inoculated into 1.3 mL LB broth. After culturing overnight at 37°C with 950 rpm shaking, 50 μL aliquots were transferred to fresh 1.3 mL LB broth and incubated at 37°C for 2 hours. The cultures received 35 µL 10% L-arabinose solution to induce RecET recombinase expression for 40 minutes. Cells underwent centrifugation at 9,600 g for 1 min at room temperature (RT), with removal of supernatant followed by two successive washes using 1 mL sterile distilled water. After the final centrifugation, 970 µL supernatant was removed and discarded, leaving concentrated cells for subsequent transformation procedures.
To construct plasmid pBBR1-PRha-Redγβα-xseB-PBAD-Cas9-xseAi-Km via RecET-mediated linear plus linear homologous recombination (LLHR), electroporate competent E. coli GB05-dir into a 1 mm electroporation cuvette using a mixture containing SacI-digested pBBR1-PRha-Redγβα-Km-amp-ccdB fragment, xseB gene, BGI-synthesized xseAi cassette, and BAD-Cas9 cassette. Apply a 1,350 V pulse for electroporation. Immediately add 1 mL antibiotic-free LB broth to the cuvette, transfer the suspension to a sterile 1.5 mL microcentrifuge tube, and incubate at 37 °C for 1 h. Centrifuge the cells at 9600 g for 30 s at RT, and discard 950 µL supernatant. Resuspend pelleted cells in the remaining 30 μL medium and plated onto LB agar containing 15 μg mL−1 kanamycin using a sterile loop. Incubate the plates at 37 °C for 24 hours. Construct plasmids pBBR1-PRha-BAS-xseBkt2440-PBAD-Cas9-xseAi-Km and pBBR1-PRha-Redγ-Redαβ7029-xseB7029-Km following identical LLHR protocols, substituting strain-specific components.
After colony growth, select 12 colonies from the plate and streak onto fresh plates for preservation, then inoculate into 1.8 ml of antibiotic-supplemented LB broth and incubate at 37°C for 12 hours. Plasmid extraction followed standard protocols. Isolated expression plasmids underwent verification through restriction enzyme digestion and sequencing. Correct plasmids were preserved in water at −20°C.
The biosensor plasmid uses an RK2 origin of replication (oriV), with gfp and gentamicin resistance genes, regulated by the J233 promoter and an E. coli codon-optimized fapR gene. For plasmid assembly, amplify oriV from plasmid RK2-apra-cm using primers rk2-1/2; amplify the J233-GFP cassette from plasmid pBBR1-Rha-GFP-kan with primers J233-GFP-1/2. Extract the gentamicin resistance gene from plasmid R6K-loxM-genta with primers genta-1/2; amplify the ampicillin resistance gene from plasmid R6K-amp-ccdB with primers amp-1/2. The fapRecoli gene was synthesized by BGI. Co-transform these five fragments into E. coli GB05-dir for LLHR according to Steps 1.5-1.6, generating plasmid RK2-PfapO-GFP-genta-FapRecoli-amp. Isolate plasmid as described in Step 1.7, is validated through restriction enzyme digestion and sequencing. Construct plasmids RK2-PfapO-GFP-genta-FapR7029-amp and RK2-PfapO-GFP-genta-FapRkt2440-amp using identical methods, replacing fapRecoli with fapR genes for S. brevitalea DSM7029- and P. putida KT2440-optimized fapR variants respectively.
Generate the gRNA cassette by mutually primed PCR synthesis using phosphorothioate-modified primers. One primer contains the universal tracrRNA sequence, and the other incorporates the target-specific gene sequence.
Generate recombineering substrates via PCR amplification with phosphorylated/phosphorothioate-modified 5’ terminal primers to enhance dsDNA stability, improve ReaL-MGE efficiency, and facilitate homology arm integration for precise gene targeting.
Part 2
Establishment of ReaL-MGE
Electroporation of expression plasmids into the E. coli BL21, S. brevitalea DSM7029 and P. putida KT2440
Puncture the lid of a 1.5 mL microcentrifuge tube using a syringe needle (25-gauge 5/8, 0.5 mm × 16 mm). Add 1.3 mL of antibiotic-free LB medium into the tube. Transfer a single E. coli BL21 colony into the medium and culture the suspension overnight at 37°C with shaking at 950 rpm in an Eppendorf thermomixer.
Puncture the lid of a 1.5 mL microcentrifuge tube. Inoculate 1.3 mL of fresh LB medium with 50 μL of overnight culture. Incubate the culture at 37°C with shaking at 950 rpm in an Eppendorf thermomixer, monitoring OD600 until it reaches 0.8.
Centrifuge the culture at 9,600 g for 1 min at RT. Discarded the supernatant by decantation and resuspend the pellet in 1 ml of autoclaved ddH2O. Repeated the entire process twice.
Gently remove the supernatant by pipetting and discard, leaving 30 μL of ddH2O for cell resuspension.
Pipette 4 μL (500 ng) of the expression plasmid (pBBR1-PRha-Redγβα-xseB-PBAD-Cas9-xseAi-Km) and the biosensor plasmid (RK2-PfapO-GFP-genta-FapRecoli-amp) into the tube. Mix the plasmids and bacterial cells by gentle pipetting, then transfer the mixture into a 1 mm electroporation cuvette. Perform electroporation at 1350 V using an Eppendorf 2510 electroporator.
Immediately after electroporation, aseptically add 1 mL of antibiotic-free LB medium to the electroporation cuvette. Resuspend cells by gently pipetting, then transfer the mixture to a 1.5 mL microcentrifuge tube. Incubate at 37°C for 1 hour with shaking at 950 rpm in an Eppendorf thermomixer.
Spread 50 μL of recovery culture onto an LB agar plate containing kanamycin and ampicillin with a sterile 10 μL loop. Air-dry the plate in a sterile hood.
Incubate plates at 37°C for 12 hours until colony visibility.
The transfer of expression and biosensor plasmids into P. putida and S. brevitalea follow the the procedure described in Steps 2.1-2.8, with two differences: incubate plates at 30°C for 48 hours in CYMG medium for S. brevitalea, and incubate plates at 30°C for 12 hours for P. putida.
Prepare 1.5 mL microcentrifuge tubes with vented caps and add 1.3 mL of LB or CYMG medium supplemented with kanamycin and ampicillin.
Transfer single colonies from the plates into individual tubes with sterile 200 μL pipette tips, then shake cultures overnight at 30°C or 37°C at 950 rpm in an Eppendorf thermomixer.
Confirm expression and biosensor plasmids integration in selected colonies by colony PCR screening.
Part 3
multiplex malonyl-CoA metabolic and genome engineering in E. coli
ReaL-MGE 1.0 functions optimally in bacterial strains tolerating the CRISPR/Cas9 system cytotoxicity, including model organisms like E. coli and P. putida. Perform two sequential electroporations: deliver genome-editing elements in the first step, then apply counter-selection in the second step to enhance editing accuracy and efficiency.
Puncture a 1.5 mL microcentrifuge tube cap with a syringe needle (25-gauge 5/8, 0.5 mm × 16 mm). Add 1.3 mL of LB medium containing kanamycin and ampicillin. Inoculate with a single E. coli BL21 colony (harboring pBBR1-PRha-Redγβα-xseB-PBAD-Cas9-Km and RK2-J233-GFP-genta-FapRecoli-amp) and incubate overnight at 37°C with 950 rpm shaking in an Eppendorf thermomixer.
Puncture a 1.5 mL microcentrifuge tube lid. Transfer 50 μL overnight culture in 1.3 mL of LB medium containing kanamycin and ampicillin and incubate at 37°C with 950 rpm shaking in an Eppendorf thermomixer. At 40 min before optimal transformation, add 35 μL of 10% L-rhamnose to induce recombinase and XseB overexpression. Continue incubation until cells reach optimal transformation. Harvest mid-log-phase cells, wash twice with sterile water to remove residual salts, and concentrate.
Generate the gRNA cassette and dsDNA donor substrates (HAL-T7-HAR, HAL-LVA-HAR, and HAL-noncoding-HAR) following Steps 1.9-1.10. Amplify tandem PCR products using the gRNA cassette and dsDNA donor substrate as templates with primers containing the gRNA target sequences as partial homology arms.
Mix E. coli BL21 electrocompetent cells with the ligated gRNA cassette and dsDNA donor substrates (gRNA cassette-T7, gRNA cassette-LVA, gRNA cassette-nocoding). Electroporate the mixture in 1 mm gap width cuvettes at 1350 V using an Eppendorf 2510 electroporator.
Following electrotransformation, incubate the cells in 1 mL of antibiotic-free LB broth supplemented with 10 nM dNTPs (GC content is 50%) at 30°C, 950 rpm shaking for 4 hours. Introduce 35 μL of 10% L-arabinose during recovery to induce Cas9 expression.
Prepare competent cells following the first round of electroporation. Mix the cells with the gRNA cassette and perform the second electroporation.
Incubate electroporated cells in 1 mL of antibiotic-free LB broth at 30°C with 950 rpm shaking for 8 hours. Add 35 μL of 10% L-arabinose during the second recovery phase to induce Cas9 counter-selection. Adjust recovery phase parameters to balance repair efficiency and cell viability.
Part 4
FACS sorting for GFP expression from the FapR biosensor in E. coli BL21 wild type and recombinants
Develop a screening strategy for mutants exhibiting enhanced malonyl-CoA production. Construct a malonyl-CoA biosensor system using green fluorescent protein (GFP). Integrate a malonyl-CoA-responsive regulatory element into the biosensor to directly correlate intracellular malonyl-CoA levels with GFP fluorescence intensity. Analyze fluorescence of single cells using flow cytometry to quantify GFP intensity. Select candidates exhibiting the highest GFP signals, indicative of elevated malonyl-CoA levels. Set detection thresholds using control samples to reduce false positives.
Centrifuge the cultured cells from Step 3.7 at 9000 g for 1 minute at RT and remove the supernatant with a pipette carefully. Resuspend the pellet in 1 mL of sterile PBS through gentle pipetting. Repeat centrifugation and washing were to eliminate residual media components and reduce fluorescence background interference. Resuspend cells in 500 uL sterile PBS and adjust to optimal density.
Activate low-flow-rate mode to optimize sorting efficiency. Configure the FITC channel (488 nm excitation, 530/30 nm emission) for sorting GFP expression cells in E. coli BL21 wild type and recombinants using the FapR biosensor.
Load the prepared bacterial suspension into a flow cytometer-compatible tube. Analyze the sample in setup mode and optimize Forward Scatter (FSC) and Side Scatter (SSC) gain voltages to encompass all bacterial events.
Resolve bacterial populations by plotting FSC area (FSC-A) versus FSC height (FSC-H) toexclude duplicates. Generate collection plots with GFP fluorescence histograms for high GFP-expressing mutants and wild-type strains. Acquire the sample in setup mode and calibrate the acquisition channel voltage.
Acquire data from a minimum of 10,000 events and record full datasets. Define sort gate boundaries and acquisition modality for cell sorting. Load the collection tube and commence sorting.
Isolate highly efficient candidates sorted via flow cytometry and plate onto gentamicin-supplemented LB agar plates to obtain single colonies.
Prepare 1.5 mL microcentrifuge tubes with pierced caps and add 1.3 mL of LB broth supplemented with kanamycin, ampicillin, and gentamicin.
Inoculate colonies from plates into tubes using a 200 μL pipette tip (one colony per tube) and incubate overnight at 37°C with 950 rpm shaking in an Eppendorf thermomixer.
Puncture a 1.5 mL microcentrifuge tube lids and inoculate 50 μL of overnight culture into 1.3 mL of LB broth with kanamycin, ampicillin, and gentamicin. Incubate at 37°C with 950 rpm shaking in an Eppendorf thermomixer until OD600 reaches 0.8.
Transfer 200 µL bacterial culture into a black-walled clear-bottomed 96-well fluorescence microplate and include at least 3 replicates.
Perform the assay using a fluorescence enzyme marker with excitation at 488 nm and emission at 510 nm. Measure OD600 using the same sample to normalize fluorescence intensity. Calculate the GFP fluorescence/OD600 ratio to eliminate cell density effects.
Puncture 2 mL microcentrifuge tube lids and inoculate 50 μL overnight culture from Step 4.8 into 1.75 mL of LB broth with kanamycin, ampicillin, and gentamicin. Incubate at 37°C with 950 rpm shaking in an Eppendorf thermomixer until OD600 reaches 0.4.
Chill the culture on ice and centrifuged at 9000 g for 15min at 4°C. Resuspend the pellet in PBS after washing, add 120 µL of lysis buffer (45:45:10 acetonitrile: methanol: water containing 0.1 M formic acid) on ice, and vortex vigorously. Incubate the extract on ice with intermittent vortexing for 15 min.
Add ammonium hydroxide to neutralize the acetic acid and centrifuge at 15000 g for 3 min at 4°C. Analyze the supernatant by LCMS. Perform standard analysis of prepared samples using an LCDAD system coupled to a Bruker Impact HD microTOF Q III ESI-MS ion trap instrument in positive ionization mode.
Use a Thermo‱ Acclaim‱ RSLC 120 C18 column (100 × 2.1 mm, 2.2 μm particle size). Apply a solvent gradient with solvent A (10 mM tributylamine, 15 mM acetic acid, and 5% methanol in distilled water) and solvent B (isopropyl alcohol). Record detection using both the diode array and ESI-MS.
Prepare a series of malonyl-CoA standard solutions at varying concentrations. Dilute each solution with an equal volume of lysis buffer (45:45:10=acetonitrile: methanol: water containing 0.1 M formic acid). Inject 5 μL standard solution and detect the target ion (m/z = 854.12 [M+H]+). Record the peak area and generate a standard curve by plotting concentration versus peak area.
Calculate the sample peak area, substitute it into the standard curve equation to determine the malonyl-CoA concentration of the samples, and normalize it to bacterial solution concentration per unit OD600.
Confirm the correlation between fluorescence intensity and intracellular malonyl-CoA concentration by measuring GFP fluorescence intensity and malonyl-CoA content in the same strain.
Detect genotypes of mutants with the highest intracellular malonyl-CoA concentrations and identify key genes and genome reduction strategies using colony PCR. Perform whole-genome sequencing on the top candidates to verify intended mutations and exclude off-target effects. Correlate specific genomic changes with phenotypic outcomes through sequencing data analysis.
Part 5
application to different hosts
ReaL-MGE 2.0 employs a linearized CRISPR/Cas9 system to regulate virulence in Cas9-intolerant bacteria, such as S. brevitalea DSM7029. Apply ReaL-MGE 2.0 systematically to target genes related to malonyl-CoA metabolism-related genes in S. brevitalea DSM7029. The procedure involves the following detailed steps.
Prepare S. brevitalea DSM7029Δglb strains harboring pBBR1-PRha-Redγ-Redαβ7029-xseB7029-Km and RK2-PfapO-GFP-genta-FapR7029-amp following Steps 3.1-3.2. Culture at 30°C in CYMG broth containing kanamycin and ampicillin with 950 rpm shaking in an Eppendorf thermomixer.
Prepare recombineering substrates by PCR using phosphorylated or phosphorothioate-modified primers.
The linearized CRISPR/Cas9 system, comprising Cas9 and single-gRNA, was prepared by PCR. The system featured Cas9 under the Pgenta promoter and the gRNA cassette driven by the PJ23119 promoter. The amplification from the p15A-PJ23119-Cas9-cm plasmid template employs primer set Pgenta-Cas9-1/tracrRNA-Cas9-2, incorporating phosphorothioate modifications.
Mix competent S. brevitalea DSM7029Δglb cells with the Cas9+gRNA mixture and dsDNA donor substrates for targeted gene editing. Perform the first electroporation with 1 mm gap-width cuvettes at 1350 V in an Eppendorf 2510 electroporator.
Following electrotransformation, incubate cells in 1.3 mL of antibiotic-free CYMG broth supplemented with 10 nM dNTPs (GC content is 50%) at 22°C, 950 rpm shaking for 8 hours.
Prepare competent cells following the first round of electroporation. Mix the cells with the Cas9+gRNA mixture and perform the second electroporation.
After the second electroporation, cells were cultured in 1 mL of antibiotic-free CYMG broth at 22°C, 950 rpm shaking for 12 hours.
Malonyl-CoA detection and key gene identification follow the same procedures established for E. coli BL21.
The mutant library for the malonyl-CoA metabolic network in P. putida KT2440 adopted methodologies analogous to those in E. coli BL21, with a difference: after the first electroporation, incubate electroporated cells in 1.3 mL of antibiotic-free LB broth supplemented with 10 nM dNTPs (GC content is 50%) at 22°C, 950 rpm shaking for 4 hours. Following the second round of electroporation, incubate cells for 8 hours. Malonyl-CoA detection and key gene identification follow the same approach as in E. coli BL21.
Protocol references
1. Huo, L. et al. Heterologous expression of bacterial natural product biosynthetic pathways. Nat Prod Rep 36, 1412-1436 (2019).
2. Cao, M.F., Tran & Zhao, H.M. Unlocking nature's biosynthetic potential by directed genome evolution. Current Opinion in Biotechnology 66, 95-104 (2020).
3. Oyetunde, T., Bao, F.S., Chen, J.W., Martin, H.G. & Tang, Y.J.J. Leveraging knowledge engineering and machine learning for microbial bio-manufacturing. Biotechnology Advances 36, 1308-1315 (2018).
4. Gu, C.D., Kim, G.B., Kim, W.J., Kim, H.U. & Lee, S.Y. Current status and applications of genome-scale metabolic models. Genome Biology 20, 121 (2019).
5. Lawson, C.E. et al. Machine learning for metabolic engineering: A review. Metabolic Engineering 63, 34-60 (2021).
6. Csorgo, B., Nyerges, A. & Pal, C. Targeted mutagenesis of multiple chromosomal regions in microbes. Current Opinion in Microbiology 57, 22-30 (2020).
7. Ciaccia, P.N., Liang, Z., Schweitzer, A.Y., Metzner, E. & Isaacs, F.J. Enhanced eMAGE applied to identify genetic factors of nuclear hormone receptor dysfunction via combinatorial gene editing. Nat Commun 15, 5218 (2024).
8. Li, R., Li, A., Zhang, Y. & Fu, J. The emerging role of recombineering in microbiology. Eng Microbiol 3, 100097 (2023).
9. Gao, H. et al. Recent advances in genome-scale engineering in Escherichia coli and their applications. Eng Microbiol 4, 100115 (2024).
10. Lim, X., Zhang, C. & Chen, X. Advances and applications of CRISPR/Cas-mediated interference in Escherichia coli. Eng Microbiol 4, 100123 (2024).
11. Zhang, Y.M., Buchholz, F., Muyrers, J.P.P. & Stewart, A.F. A new logic for DNA engineering using recombination in Escherichia coli. Nature Genetics 20, 123-128 (1998).
12. Muyrers, J.P., Zhang, Y., Testa, G. & Stewart, A.F. Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res 27, 1555-1557 (1999).
13. Zhang, Y.M., Muyrers, J.P.P., Testa, G. & Stewart, A.F. DNA cloning by homologous recombination in Escherichia coli. Nature Biotechnology 18, 1314-1317 (2000).
14. Wang, H.H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894-898 (2009).
15. Warner, J.R., Reeder, P.J., Karimpour-Fard, A., Woodruff, L.B.A. & Gill, R.T. Rapid profiling of a microbial genome using mixtures of barcoded oligonucleotides. Nature Biotechnology 28, 856-862 (2010).
16. Wang, H.H. et al. Genome-scale promoter engineering by coselection MAGE. Nature Methods 9, 591-593 (2012).
17. Nyerges, A. et al. A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species. Proc Natl Acad Sci U S A 113, 2502-2507 (2016).
18. Nyerges, A. et al. Directed evolution of multiple genomic loci allows the prediction of antibiotic resistance. Proc Natl Acad Sci U S A 115, E5726-E5735 (2018).
19. Wannier, T.M. et al. Recombineering and MAGE. Nat Rev Methods Primers 1, 7 (2021).
20. Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L.A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31, 233-239 (2013).
21. Garst, A.D. et al. Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering. Nature Biotechnology 35, 48-55 (2017).
22. Reisch, C.R. & Prather, K.L.J. Scarless Cas9 Assisted Recombineering (no-SCAR) in Escherichia coli, an Easy-to-Use System for Genome Editing. Curr Protoc Mol Biol 117, 1-20 (2017).
23. Rostain, W. et al. Cas9 off-target binding to the promoter of bacterial genes leads to silencing and toxicity. Nucleic Acids Research 51, 3485-3496 (2023).
24. Zhang, Y. et al. A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae. Nat Commun 10, 1053 (2019).
25. Sharda, M., Badrinarayanan, A. & Seshasayee, A.S.N. Evolutionary and Comparative Analysis of Bacterial Nonhomologous End Joining Repair. Genome Biology and Evolution 12, 2450-2466 (2020).
26. Zheng, W. et al. ReaL-MGE is a tool for enhanced multiplex genome engineering and application to malonyl-CoA anabolism. Nat Commun 15, 9790 (2024).
27. Xu, P., Qiao, K., Ahn, W.S. & Stephanopoulos, G. Engineering Yarrowia lipolytica as a platform for synthesis of drop-in transportation fuels and oleochemicals. Proc Natl Acad Sci U S A 113, 10848-10853 (2016).
28. Yan, D. et al. Repurposing type III polyketide synthase as a malonyl-CoA biosensor for metabolic engineering in bacteria. Proc Natl Acad Sci U S A 115, 9835-9844 (2018).
29. Wang, X. et al. Improved dsDNA recombineering enables versatile multiplex genome engineering of kilobase-scale sequences in diverse bacteria. Nucleic Acids Res 50, e15 (2021).
30. Wang, X. et al. Discovery of recombinases enables genome mining of cryptic biosynthetic gene clusters in Burkholderiales species. Proc Natl Acad Sci U S A 115, E4255-E4263 (2018).
31. Davis, J.R. et al. Efficient in vivo base editing via single adeno-associated viruses with size-optimized genomes encoding compact adenine base editors. Nat Biomed Eng 6, 1272-1283 (2022).
32. Gonzalez-Delgado, A., Lopez, S.C., Rojas-Montero, M., Fishman, C.B. & Shipman, S.L. Simultaneous multi-site editing of individual genomes using retron arrays. Nat Chem Biol (2024).
33. Isaacs, F.J. et al. Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science 333, 348-353 (2011).
34. Carr, P.A. et al. Enhanced multiplex genome engineering through co-operative oligonucleotide co-selection. Nucleic Acids Research 40 (2012).
35. Dalia, A.B., McDonough, E. & Camilli, A. Multiplex genome editing by natural transformation. Proc Natl Acad Sci U S A111, 8937-8942 (2014).
36. Wang, K. et al. Defining synonymous codon compression schemes by genome recoding. Nature 539, 59-64 (2016).
37. Zhang, Y. et al. Multicopy Chromosomal Integration Using CRISPR-Associated Transposases. ACS Synth Biol 9, 1998-2008 (2020).
38. Gaudelli, N.M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464-471 (2017).
39. Anzalone, A.V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149-157 (2019).
40. Wang, Y. et al. In-situ generation of large numbers of genetic combinations for metabolic reprogramming via CRISPR-guided base editing. Nat Commun 12, 678 (2021).
Acknowledgements
This work was supported by the National Key R&D Program of China (No. 2023YFC3402000), the National Natural Science Foundation of Shandong province (ZR2023YQ028), Taishan Scholar Program of Shandong Province in China (tsqn202312007), and SKLMT Frontiers and Challenges Project of Shandong University.