TITLE: Determining the horizontal transfer of antibiotic resistance genes: using high-throughput fluorescence-based sorting approaches
AUTHORS: Qiu E Yang, Yanshuang Yu

[DESCRIPTION]
Despite the significant role of plasmids play in the global spread of antimicrobial resistance (AMR), current methods to study the transfer of antibiotic resistance genes (ARGs) mainly rely on bacterial cultivation or sequencing techniques, leaving a knowledge gap in understanding ARGs dissemination transfer within natural microbial communities. To address this, new tools allowing real-time tracking of the spread of ARGs are essential for comprehensive environmental risk assessments. Herein, we present a culture-independent protocol for examining the horizontal transfer of ARGs across diverse bacterial populations. This method utilizes CRISPR-based editing to fluorescently label wild-type AMR plasmids, facilitating their identification and sorting via Fluorescence-activated cell sorting (FACS). As an illustrative example, we detail a step-by-step protocol targeting gfp-tagged AMR plasmid, followed by conjugation procedures and FACS-based selection of green fluorescent protein (GFP)-expressing transconjugants. This fluorescence-based approach applied to real-life bacterial populations can be uniquely deployed to examine One Health risk factors such climate changes and environmental pollution.

[STEPS]
SECTION: Molecular cloning of plasmid vector pSL1521::gfp (4-5 days)
1. Plasmid extraction: extract plasmid DNA from up to 5 mL of overnight culture of E. coli DH5α strain containing the pSL1521 plasmid (Addgene, 160729) via a TIANprep Mini Plasmid Kit (TIANGEN, DP103-02). Measure plasmid DNA concentration using the Qubit Flex Fluorometer (Invitrogen, Q33326).

SECTION: Molecular cloning of plasmid vector pSL1521::gfp (4-5 days)
6. Transformation:
(i) Mix 5 µL of the ligation reaction from step 5 with the competent E. coli DH5α (TAKARA, 9057) and incubate on ice for 30 min.
(ii) Heat-shock the mixture for 45 s at 42 °C on a Thermo Shaker Incubator (MIULAB, MTH-100), followed by 2 min incubation on ice.
(iii) Add 900 µL of prepared LB medium (30 °C) and recover the cells for 60 min at   30 °C with agitation at  .
(iv) Plate 100 µL of the transformed culture on selective LB plates supplemented with 50 mg/L of spectinomycin.
SECTION: Molecular cloning of plasmid vector pSL1521::gfp (4-5 days)
3. Gel purification of digested plasmid DNA: run the digested plasmid on 1% (wt/vol) agarose gel, excise the band corresponding to the plasmid backbone (~5 kb) and purify it using the Gel Extraction Kit (TIANGEN, China) according to the manufacturer’s instructions.
 


SECTION: Molecular cloning of plasmid vector pSL1521::gfp (4-5 days)
2. Vector digestion: digest the plasmid pSL1521 with XhoI (NEB, R0146S) and PstI (NEB, R0140S)  in a 50 µL reaction containing the following components:
 
Incubate the reaction at 37 °C for 15 min~30 min, followed by an inactivation step at 65 °C for 20 min.

SECTION: Molecular cloning of plasmid vector pSL1521::gfp (4-5 days)
7. Verification of transformants:
(i) Pick several single clones from step 6 and make cell suspensions as DNA templates, perform a cPCR in a 15 µL reaction volume containing the following components:
 
 
(ii) Run the PCR with the following program:
 
 
(iii) Perform gel electrophoresis to confirm amplicon size, followed by Sanger sequencing.
(iv) Keep pSL1521::gfp-positive strains in 20 % (v/v) glycerol and store at -80 °C for future investigation. 

SECTION: Spacer cloning of pSL1521::gfp::spacer (3 days)
8. Spacer design: select a 32 bp genomic sequence immediately preceding the 5′CC PAM, and add the overhang nucleotides to the forward and reverse spacer  oligonucleotides to allow cloning into the BsaI of the pSL1521::gfp plasmid.
 
 

SECTION: Spacer cloning of pSL1521::gfp::spacer (3 days)
11. Purify the digested pSL1521::gfp using the Gel Extraction Kit (refer to step 3).

 

SECTION: Spacer cloning of pSL1521::gfp::spacer (3 days)
9. Phosphorylation and annealing of the complementary oligonucleotides:
(i) Mix 1 µL of each oligo pair with T4 Polynucleotide Kinase (NEB, M0201S) as following mixture and incubate at37 °C for 30 min: 
 
        
(ii) Denature the primers at 95 °C for 5 min and allow the mixture to cool down to Room temperature (25 °C) using a thermocycler (BIO-RAD, CT062680).
        
 

SECTION: Spacer cloning of pSL1521::gfp::spacer (3 days)
10. Vector digestion: Digest vector pSL1521::gfp with BsaI (NEB, R3733S) in a 50 µL reaction containing the following components:
 
Incubate for 60 min at 37 °C, followed by inactivation at 65 °C for 20 min.

SECTION: Spacer cloning of pSL1521::gfp::spacer (3 days)
12. Ligate the phosphorylated dsDNA spacer fragment into the BsaI-digested pSL1521::gfp from step 10-11 using T4 DNA Ligase (NEB, M0202S) as follows. 
 
 Incubate the reaction at 22 °C ~ 25 °C for 60 min, followed by heat inactivation at 72 °C for 20 min.

SECTION: Spacer cloning of pSL1521::gfp::spacer (3 days)
13. Transformation: mix 10 µL of the ligation reaction (from step 12) with chemically competent E. coli DH5α cells, follow the remaining steps of the transformation procedure detailed in step 6.

SECTION: Spacer cloning of pSL1521::gfp::spacer (3 days)
14. Perform cPCR and sanger sequencing to confirm insertion of target spacer in pSL1521::gfp, following a procedure similar to step 7.

SECTION: Introducing a gfp tagging into a wild-type plasmid (3 ~ 5 days)
15. Preparation of electronically competent cells:
(i) Grow overnight culture of E.coli MG1655 containing a desire AMR plasmid in 10 mL of LB broth with appropriate antibiotics.
(ii) Inoculate 1 mL of the 20 min culture into a 500 mL Erlenmeyer flask containing 100 mLof LB medium and incubate for 3 ~4 h at 37 °C wi until the OD600nm=0.5~0.6.
(iii) Harvest and centrifuge the cultures at   and discard  the supernatant.
(iv) Resuspend cell pellet in ice-cold ddH2O and centrifuge at  . Discard the supernatant and repeat this step once.
(v) Resuspend cell pellet in ice-cold 10% (wt/vol) glycerol and  centrifuge at  . Discard the supernatant and repeat this step once.
(vi) Prepare 100 µLaliquots in 1.5 mL tubes.

SECTION: Introducing a gfp tagging into a wild-type plasmid (3 ~ 5 days)
16. Electroporation:
(i) Mix approximately 500 ng of pSL1521::gfp::spacer (prepared in step 15)  with 100 µLof electronically competent cells (prepared in step 16).
(ii) Transfer bacterial-plasmid mixture into a 0.1 cm electroporation cuvette (BIO-RAD, 1652083), and apply an electric pulse using the MicroPulser eletroporator (Bio-Rad, program EC1, 1.8 kV).
(iii) Immediately add 900 µL LB broth into the cuvette, mix gently, transfer to a  sterile 1.5 mL tube, and incubate at 37 °Cwith constant agitation ( )
SECTION: Introducing a gfp tagging into a wild-type plasmid (3 ~ 5 days)
17. Plate bacterial culture on selective agar plates similar to step 6.
SECTION: Introducing a gfp tagging into a wild-type plasmid (3 ~ 5 days)
18. Perform cPCR verification of successfully gfp-tagged AMR plasmid following step 7, followed by sanger sequencing.

SECTION: Elimination of pSL1521::gfp::spacer from bacterial strains (2~4 days)
19. Streak the transformants (from step 19) onto an LB agar with appropriate antibiotics, and incubate overnight at 37 °C.
SECTION: Elimination of pSL1521::gfp::spacer from bacterial strains (2~4 days)
20. Pick a colony and streak onto fresh plate and incubate at 37 °Cfor 1440 min. Repeat this plasmid curing passages until the loss of plasmid pSL1521::gfp::spacer.
SECTION: Elimination of pSL1521::gfp::spacer from bacterial strains (2~4 days)
22. Keep bacterial cultures with gfp-tagged AMR plasmid in 20 % (v/v) glycerol and store at -80 °C for future investigation.

SECTION: Conjugation procedures (1 day)
23.  Mix donor culture and recipient community with the ratio of 1:1 (v/v), and co-incubate for 16-20 h at 37 °C.

 

SECTION: Perform the Fluorescence-activated cell sorting (FACS) (1~2 days)
25. Sample preparation: dilute the mating culture in PBS buffer to ~8000 counting events per second to assure for optimal sorting.

 

SECTION: Perform the Fluorescence-activated cell sorting (FACS) (1~2 days)
26. Sorting speed: set sorting speed to less than 10,000 events per second. Avoid excessively high sorting speed to prevent sorting of adhesive cells.
SECTION: Perform the Fluorescence-activated cell sorting (FACS) (1~2 days)
27. Sort gfp-positive cells initially and perform a second round of sorting to ensure sorting purity. 

 

SECTION: Molecular cloning of plasmid vector pSL1521::gfp (4-5 days)
4. Preparation of gfp DNA fragment:
(i) PCR amplify gfp gene from a gfp-positive template plasmid using primers containing XhoI and PstI overhang nucleotides as follows:
 
 
(ii) Run PCR according to the following program:
 
 
SECTION: Molecular cloning of plasmid vector pSL1521::gfp (4-5 days)
5. Digestion and ligation:
(i) Digest the gfp amplicon with XhoI and PstI restriction enzymes as step 2. 
(ii) Ligate the digested gfp fragment and the purified plasmid from step3 using the NEBuilder ® HiFi DNA Assembly Master Mix (NEB, E5520S) as follows:
 
 
 
(iii) Incubate the reaction at50 °C for 30 min.

SECTION: Perform the Fluorescence-activated cell sorting (FACS) (1~2 days)
24. Perform preliminary experiments using gfp-positive, mCherry-positive and fluorescence-negative controls, to optimize forward and side scatter threshold and gate settings.
SECTION: Perform the Fluorescence-activated cell sorting (FACS) (1~2 days)
28.  After sorting, plate the sorted cells on selective agar plates and verify by cPCR and sanger sequencing.
SECTION: Elimination of pSL1521::gfp::spacer from bacterial strains (2~4 days)
21. Perform cPCR to verify the loss of pSL1521::gfp::spacer plasmid, similar to step 7.