Determining the horizontal transfer of antibiotic resistance genes: using high-throughput fluorescence-based sorting approaches

Plasmid extraction : extract plasmid DNA from up to 5mL 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).

Vector digestion : digest the plasmid pSL1521 with XhoI (NEB, R0146S) and PstI (NEB, R0140S) in a 50µL reaction containing the following components:

A	B	C
Component	Amount (µL)	Final concentration
pSL1521	up to 1 ug	500 ng/µL
XhoI	1
PstI	1
rCutSmart™ Buffer	5
Nuclease-free Water	adjust to 50 µL final volume
Total volume	50

Incubate the reaction at 37°C for 0h 15m 0s~0h 30m 0s, followed by an inactivation step at 65°C for 0h 20m 0s.

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.

Preparation of gfp DNA fragment: gfp DNA fragment:

(i) PCR amplify gfp gene from a gfp- positive template plasmid using primers containing XhoI and PstI overhang nucleotides as follows:

A	B	C
Component	Amount (µL)	Final concentration
Forward primer	1	0.3 µM
Reverse primer	1	0.3 µM
2× Phanta Flash Master Mix (Dye Plus)	12.5	1×
Template	1
Nuclease-free Water	9.5
Total volume	25

(ii) Run PCR according to the following program:

A	B	C	D	E
Cycle no.	Denaturation	Annealing	Extension	Final
1	98 °C, 30 s
2-31	98 °C, 10 s	58 °C, 5 s	72 °C, 20 s
32			72 °C, 1 min
33				4 °C, hold

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:

A	B	C
Component	Amount (µL)	Final concentration
HiFi DNA Assembly Master Mix	10	1 x
Digested pSL1521 plasmid	up to 100 ng	50-100 ng
Insert gfp fragment	up to 200 ng	100-200 ng
Nuclease-free Water	adjust to 20 µL final volume

(iii) Incubate the reaction at50°C for 0h 30m 0s.

Transformation :

(i) Mix 5µL of the ligation reaction from step 5 with the competent E. coli DH5α (TAKARA, 9057) and incubate On ice for 0h 30m 0s.

(ii) Heat-shock the mixture for 0h 0m 45s at 42°C on a Thermo Shaker Incubator (MIULAB, MTH-100), followed by 0h 2m 0s incubation On ice.

(iii)  Add 900µL of prepared LB medium (30°C) and recover the cells for 1h 0m 0s at   30°C with agitation at 180rpm.

(iv) Plate 100µL of the transformed culture on selective LB plates supplemented with 50mg/L of spectinomycin.

Verification of transformants :

(i) Pick several single clones from step 6 and make cell suspensions as DNA templates, perform a cPCR in a 15µL reaction volume containing the following components:

A	B	C
Component	Amount (µL)	Final concentration
Forward primer	0.5	0.3 µM
Reverse primer	0.5	0.3 µM
2× SanTaq PCR Master Mix	7.5	1×
Template (cell suspension)	1
Nuclease-free Water	5.5
Total volume	15

(ii) Run the PCR with the following program:

A	B	C	D	E
Cycle no.	Denaturation	Annealing	Extension	Final
1	94 °C, 5 min
2-31	94 °C, 30 s	58 °C, 30 s	72 °C, 1 min
32			72 °C, 5 min
33				4 °C, hold

 

(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.

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.

 

Phosphorylation and annealing of the complementary oligonucleotides :

(i) Mix 1µL of each oligo pair with T4 Polynucleotide Kinase (NEB, M0201S) as following mixture and incubate at37°C for 0h 30m 0s:

A	B	C
Component	Amount (µL)	Final concentration
Forward oligonucleotide (100 µM)	1	10 µM
Reverse oligonucleotide (100 µM)	1	10 µM
T4 Polynucleotide Kinase	1	1000 units/mL
T4 Polynucleotide Kinase Reaction Buffer (10×)	1	1×
Nuclease-free Water	6
Total volume	10

       

(ii) Denature the primers at 95°C for 0h 5m 0s and allow the mixture to cool down to Room temperature (25 °C) using a thermocycler (BIO-RAD, CT062680).

       

Vector digestion : Digest vector pSL1521:: gfp with BsaI (NEB, R3733S) in a 50 µL reaction containing the following components:

A	B	C
Component	Amount (µL)	Final concentration
plasmid DNA (pSL1521::gfp)	>1 (up to 500 ng)	10 ng/µL
BsaI-HF®v2 (20,000 units/mL)	0.5	200 units/mL
rCutSmart™ Buffer	5	1×
Nuclease-free Water	adjust to 50 µL final volume
Total volume	50

Incubate for 1h 0m 0s at 37°C, followed by inactivation at 65°C for 0h 20m 0s.

Purify the digested pSL1521:: gfp using the Gel Extraction Kit (refer to step 3).

Ligate the phosphorylated dsDNA spacer fragment into the BsaI-digested pSL1521:: gfp from step 10-11 using T4 DNA Ligase (NEB, M0202S) as follows.

A	B	C
Component	Amount (µL)	Final concentration
BsaI-digested pSL1521::gfp (step11)	10	10 µM
dsDNA spacer (10 µM) (step9)	2	10 µM
T4 DNA Ligase (400,000 units/mL)	0.5	1000 units/mL
T4 DNA Ligase Reaction Buffer (10×)	2	1×
Nuclease-free Water	5.5
Total volume	20

 Incubate the reaction at 22°C ~ 25°C for 1h 0m 0s, followed by heat inactivation at 72°C for 0h 20m 0s.

Transformation : mix 10µL 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.

Perform cPCR and sanger sequencing to confirm insertion of target spacer in pSL1521:: gfp , following a procedure similar to step 7.

Preparation of electronically competent cells:

(i) Grow overnight culture of E.coli MG1655 containing a desire AMR plasmid in 10mL of LB broth with appropriate antibiotics.

(ii) Inoculate 1mL of the 0h 20m 0s culture into a 500mL Erlenmeyer flask containing 100mLof LB medium and incubate for 3 ~4 h at 37°C wi180rpmuntil the OD_600nm=0.5~0.6.

(iii) Harvest and centrifuge the cultures at 5000rpm and discard the supernatant.

(iv) Resuspend cell pellet in ice-cold ddH₂O and centrifuge at 5000rpm. Discard the supernatant and repeat this step once.

(v) Resuspend cell pellet in ice-cold 10% (wt/vol) glycerol and centrifuge at 5000rpm. Discard the supernatant and repeat this step once.

(vi) Prepare 100µLaliquots in 1.5mL tubes.

Electroporation :

(i) Mix approximately 500ng of pSL1521:: gfp ::spacer (prepared in step 15) with 100µLof 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 900µL LB broth into the cuvette, mix gently, transfer to a sterile 1.5 mL tube, and incubate at 37°Cwith constant agitation (180rpm)

Plate bacterial culture on selective agar plates similar to step 6.

Perform cPCR verification of successfully gfp -tagged AMR plasmid following step 7, followed by sanger sequencing.

Streak the transformants (from step 19) onto an LB agar with appropriate antibiotics, and incubate overnight at 37°C.

Pick a colony and streak onto fresh plate and incubate at 37°Cfor 24h 0m 0s. Repeat this plasmid curing passages until the loss of plasmid pSL1521:: gfp ::spacer.

Perform cPCR to verify the loss of pSL1521:: gfp ::spacer plasmid, similar to step 7.

Keep bacterial cultures with gfp -tagged AMR plasmid in 20% (v/v) glycerol and store at -80°C for future investigation.

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

Perform preliminary experiments using gfp -positive, mCherry -positive and fluorescence-negative controls, to optimize forward and side scatter threshold and gate settings.

Sample preparation : dilute the mating culture in PBS buffer to ~8000 counting events per second to assure for optimal sorting.

Sorting speed : set sorting speed to less than 10,000 events per second. Avoid excessively high sorting speed to prevent sorting of adhesive cells.

Sort gfp -positive cells initially and perform a second round of sorting to ensure sorting purity.

 After sorting, plate the sorted cells on selective agar plates and verify by cPCR and sanger sequencing.