Jan 07, 2026
Version 3
Conjugation-Based Mutagenesis V.3
This protocol is a draft, published without a DOI.
- 1Washington University School of Medicine
Protocol Citation: Katie O'Connor 2026. Conjugation-Based Mutagenesis. protocols.io https://dx.doi.org/Version created by Katie O'Connor
Manuscript citation:
Cianfanelli, F.R., Cunrath, O. & Bumann, D. Efficient dual-negative selection for bacterial genome editing. BMC Microbiol 20, 129 (2020). https://doi.org/10.1186/s12866-020-01819-2
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: January 05, 2026
Last Modified: January 07, 2026
Protocol Integer ID: 237087
Keywords: based mutagenesi, based mutagenesis protocol, mutagenesis protocol, bmc microbiology, conjugation, mutagenesi
Abstract
This is a conjugation based mutagenesis protocol adapted from Cianfanelli et al. BMC Microbiology
Materials
Equipment:
NanoDrop One/OneC Microvolume UV-Vis Spectrophotometer, ThermoFisher cat no. ND-ONE-W
PTC Tempo 48/48 Thermal Cycler, BIO-RAD cat no. 12015309
Owl EasyCast B2 Mini Gel Electrophoresis Systems, ThermoFisher cat no. B2-BP
PowerPac Basic Power Supply, BIO-RAD cat no. 1645050
Sorvall Legend Micro 21R Microcentrifuge, ThermoFisher cat no.75002559
Non-Consumables:
Corning PYREX 1 Liter Storage Bottles, Fisher cat no. 13951L
250 mL Erlenmeyer Flask with Vented Cap, Fisher cat no. 10-040F
14 mL Glass Culture Tubes, Fisher cat no. 21-200-203
Reagent Kits:
QIAamp DNA Mini Kit, QIAGEN cat no. 51304
QIAquick PCR Purification Kit for PCR Cleanup, QIAGEN cat no. 28104
PR1MA Hot Start Taq, MIDSCI cat no. PR1001-HSR-1000
ZR Plasmid Miniprep - Classic, Zymo Research cat no. D4054
Gibson Assembly Master Mix, New England Biolabs cat no. E2611L
Plastic Consumables:
1.5 mL microcentrifuge tubes, Fisher cat no. 05-408-129
50 mL conical tubes, VWR cat no. 89039-656
15 mL conical tubes, VWR cat no. 89039-664
Petri Dishes with Clear Lid, Fisher cat no. FB0875712
MCE Membrane Filter, 0.45 μm Pore Size, Millipore cat no. HAWP04700
Chemical Consumables:
Luria Broth, RPI cat no. L24045-5000.0
200 proof ethanol, Sigma-Aldrich cat no. 4450-OP
Diamino pimelic acid (DAP), Sigma-Aldrich cat no. D1377-10G
Calcium chloride, Sigma-Aldrich cat no. C4901
Glycerol, Sigma-Aldrich cat no. G7893
Agarose, Lamda Biotech cat no. A113-3
SYBR Safe DNA Gel Stain, Invitrogen cat no. S33102
Sucrose, RPI cat no. S24060-5000.0
Tryptone, gibco cat no. 211705
Yeast, gibco cat no. 212750
Agar, BD cat no. 214030
Sodium chloride (NaCl), Sigma-Aldrich cat no. S9888
Potassium chloride (KCl), Sigma-Aldrich cat no. 793590
Anhydrous Magnesium chloride (MgCl2), Sigma-Aldrich cat no. M8266
Glucose, Sigma-Aldrich cat no. G7021
Tris Base, Sigma-Aldrich cat no. 252859
Acetic Acid, Sigma-Aldrich cat no. 695092
0.5 M EDTA, Invitrogen cat no. AM9260G
Antibiotics:
Kanamycin sulfate, Sigma-Aldrich cat no. K4000
Anhydrotetracycline, Sigma-Aldrich cat no.94664
Step 1: Design Primers
Primer Design for Assembly of Restriction Enzyme Digested Vector and PCR-Generated Insert.
Restriction enzyme-treated vectors can have 5´-overhangs, 3´-overhangs or blunt ends. When vector is linearized by restriction digestion, the entire overlap sequence must originate from the vector sequence and must be added to primers that will be used to amplify the insert.
The overlap region of the forward primer for the gene of interest (orange) should line up with the 3´ end of the overhang on the vector’s left arm and extend back until the Tm ³ 48°C (Fig.4A, Left side). This primer also includes gene-specific sequence at the 3´-end (gray).
Keep in mind that the restriction site, which was used to digest the vector, will be lost in the assembled product. However, additional nucleotides may be added between the overlap region and gene-specific sequence region to restore the pre-existing restriction site, or to introduce a new, unique restriction site. A similar principle is applied to the design of the reverse primer for the gene of interest (Fig. 4A, Right side).
Figure 4B shows primer design for assembly of the lacZ gene and pMAL-c5X, digested with NcoI and SbfI.
In this example, the forward primer of the gene has a "C" nucleotide (underlined) inserted between the 18-nt overlap and the N-terminal sequence of the lacZ gene to ensure the lacZ protein is in frame with the maltose binding protein.
Start with NEB’s Gibson assembly tool
Note
Need 700 bp upstream and downstream “flanking regions” of the gene you want to knock out into one of the suicide vectors.
Vector Options: pFOK (KAN resistant), pFOKT (KAN and Tellurite resistant), and pFOG (Gent. resistant).
Add pFOK sequence as your first “fragment”, check the boxes for “Vector” and “Circular”, and select “Restriction Digest” as your method.
Choose 2 restriction enzymes (REs).
Note
Any REs are okay, as long as there are cut sites for it in the multiple cloning region (MCR) of pFOK.
Open the plasmid in SnapGene and check sites.
Once you have everything in NEB’s Gibson tool, test out REs to achieve optimal Tm’s for primers.
Add the 700 bp upstream and downstream flanking regions each as their own fragments.
Note
Can copy and paste the sequence directly from SnapGene.
Change the “Build Settings” to the Gibson Assembly Master Mix, 15-25 nt of overlap, Taq DNA Polymerase with Standard Buffer. Keep the rest of the default settings.
Note
Shoot for ~40-45 nt total length of the primers to avoid issues with amplifying inserts.
The NEB Gibson tool should then display 4 “Required Oligonucleotides” to order so you can amplify the flanking regions via PCR.
Using the IDT DNA Oligonucleotide ordering form estimate the annealing temperatures for your primers.
Note
Upstream flanking region forward and reverse should match and the downstream flanking region forward and reverse to match (≤ 3°C).
Overlapping primers with the plasmid/vector tend to have high annealing temperatures, challenging to match up.
Trial REs to find lowest annealing temperatures for the outer primers by clicking the pen icon on the fragment.
Once “outer” primers that overlap with the plasmid are obtained, save in IDT tab and put them into SnapGene.
Note
Actions > Gibson Assembly > insert multiple fragments.
Return to NEB’s Gibson assembly tool and toggle “Minimum Primer Length” to ensure "inner" and "outer" primers match, input into IDT until Tm's are ≤ 3°C different.
Note
After matching up 4 primers, go into SnapGene’s Gibson assembly tool to ensure alignment.
Load pFOK into the Vector tab, choose linearize with REs, choose the REs you picked on the Gibson assembly tool page.
Use a Genome (ex. 43816) as your “source” of the fragment, choose "use as a template for PCR", and pick Taq.
Note
In the fragment tab switch between the 2 fragments and copy over your primers from the IDT page.
Check on "product" tab that fragments are in alignment.
Note
Your fragments should overlap with the plasmid and with each other.
You should also see a green box in the bottom right corner that says “Ready for Gibson Assembly.”
Design upstream and downstream check primers ~700 bp outside desired region to be used for final screen.
Email Jordan to order primers.
Step 2: Amplify the Insert for Target Vector
1h 52m 45s
When primers arrive, culture desired knockout strain (e.g. TOP52) AND one of the strains harboring the suicide vector you want to use (Jke201 or Ecs17) at 37 °C shaking overnight.
Note
Located in DAR Box 6.
1h
The following day genomic DNA prep the strain you want to make a knockout to use as a template with Qiagen DNA Miniprep kit. (store gDNA at -20 °C ).
Note
Heat one water bath or heating block to 56 and one to 70 degrees.
Transfer 1 mL of culture into 1.5 mL microcentrifuge tube.
Centrifuge at 21.1 x g, 00:05:00 .
5m
Discard supernatant and re-suspend in 170 µL of buffer ATL.
Add 20 µL proteinase K and mix by vortexing for 00:00:15 .
15s
Incubate at 56 °C for 00:20:00 .
20m
Add 200 µL buffer AL, mix by vortexing for 00:00:15 .
15s
Incubate at 70 °C for 00:10:00 .
10m
Add 200 µL of 200 proof ethanol, mix by vortexing for 00:00:15 .
15s
Add mixture to QIAamp Mini Spin column in a 2 mL collection tube.
Centrifuge at 6 x g, 00:01:00 .
1m
Discard flow through and place the QIAamp Mini Spin column in a fresh 2 mL collection tube.
Add 500 µL buffer AW1 and centrifuge at 6 x g, 00:01:00 .
1m
Discard flow through and place the QIAamp Mini Spin column in a fresh 2 mL collection tube.
Add 500 µL buffer AW2 and centrifuge at 21.1 x g, 00:03:00 .
3m
Discard flow through and place the QIAamp Mini Spin column in same 2 mL collection tube.
Centrifuge at 21.1 x g, 00:01:00 .
1m
Discard flow through and place the QIAamp Mini Spin column in a 1.5 mL microcentrifuge tube.
Add 50 µL of buffer AE and incubate at room temperature for 00:05:00 .
5m
Centrifuge at6 x g, 00:01:00 .
1m
Repeat steps 16.18 and 16.19 to increase yield.
PCR up the flanking regions using the following recipe:
On thermocycler, select AO check, edit step #3 for Tm, #4 for extension time.
Note
Tm = fwd + rvs/ 2.
Check IDT sheet for extension time.
Number of BP x 90 / 1000.
Purify PCR product using QIAquick PCR Purification Kit for PCR Cleanup
Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix.
Note
If the color of the mixture is orange or violet, add 10 uL 3 M sodium acetate, pH 5.0, and mix.
The color of the mixture will turn yellow.
Place a QIAquick column in a provided 2 mL collection tube.
To bind DNA, apply the sample to the QIAquick column and centrifuge at 17.9 x g, 00:01:00 .
1m
Discard flow-through and place the QIAquick column back into 2 mL collection tube.
Add 750 µL buffer PE to the QIAquick column and centrifuge at 17.9 x g, 00:01:00 to wash.
1m
Discard flow-through and place the QIAquick column back into 2 mL collection tube.
Centrifuge at 17.9 x g, 00:01:00 to remove residual wash buffer.
1m
Place each QIAquick column in a clean 1.5 mL microcentrifuge tube.
To elute DNA, add 30 µL SMQ H2O to the center of the QIAquick membrane.
Let column sit for 00:01:00 .
1m
Centrifuge at 17.9 x g, 00:01:00 .
1m
Measure DNA concentration using nanodrop.
Store at 4 °C for up to 48 hours.
Step 3: Plasmid Prep Out Suicide Vector
8m 20s
Digest plasmid using restriction enzymes selected during step 1 (ex. BamHI, EcoRi).
Note
Follow instructions in package, except incubate for 1 hour instead of 15 minutes.
Example: 1x rxn = 1 µg of plasmid, 5 µL of CutSmart buffer, 1 µL of the RE, and then H2O up to 50 µL total. Incubate at 37°C for 1 hr.
Some REs then need to be heat inactivated for 20 minutes at 65°C (find card or look it up on NEB).
Perform 8x and at least three 1 mL replicates to pool together.
Note
Pre-warm elution buffer in 37 °C incubator.
Centrifuge 1.5 mL bacterial culture in 1.5 mL microcentrifuge tube at 16 x g, 00:00:20 .
20s
Discard supernatant and resuspend in 200 µL of P1 Buffer (red).
Add 200 µL P2 Buffer (blue) and mix by inverting. Cells are completely lysed when the solution appears clear, purple, and viscous. No more than00:02:00 .
2m
Centrifuge at 16 x g, 00:02:00 .
2m
Place a Zymo-Spin column in a 2 mL collection tube and transfer supernatant into column.
Centrifuge at 16 x g, 00:00:30 .
30s
Discard flow through, making sure no liquid touches the bottom of the column. Return column to collection tube.
Add 200 µL of Endo-Wash Buffer and centrifuge at 16 x g, 00:00:30 .
Add 400 µL of Plasmid Wash Buffer to the column and 16 x g, 00:01:00
1m
Transfer column to fresh 1.5 mL microcentrifuge tube and add 30 µL warmed Elution Buffer.
Centrifuge at 16 x g, 00:00:30 to elute plasmid DNA.
30s
Record concentration using nanodrop.
Step 4: Gibson Assembly to Combine DNA fragments and Linearized Vector
1h
Vortex the master mix and then add:
Note
Example Calculation:
Upstream fragment 681.8 ng/µL, want 10 uL of
To get 75 ng/µL, plug in using C1V1=C2V2
681.8 ng/µL (x µL) = 75 ng/µL (10 µL)
X=1.1 µL
So add 1.1 µL of 681.8 ng/µL upstream fragment to 8.9 µL SMQ H2O
Mix well and briefly centrifuge.
Incubate at 50 °C for 01:00:00 in thermocycler.
1h
Store at-20 °C or proceed to transformation (recommend avoid freezing).
Step 5: Insert Linearized Vector into Chemically Competent Cells (Transformation)
1h 35m 30s
Make Chemically Competent E. coli
Note
Can use Jke201 (requires DAP to grow) or Ecs17.
Inoculate 50 mL fresh LB (+100 micromolar (µM) DAP if using Jke201) with 500 µL of overnight culture.
Wash twice with 25 mL ice-cold 100 millimolar (mM) CaCl2 solution containing 15 % volume of glycerol.
Resuspend in 5 mL ice-cold 100 millimolar (mM) CaCl2 solution containing 15 % volume of glycerol.
Aliquot 200 µL into eppie tubes and store at -80 °C .
Add 5 µL of Gibson assembly reaction mixture to 100 µL aliquot of chemically competent cells.
Gently invert to mix and incubate on ice for 00:30:00 .
Heat shock 00:00:30 42 °C followed by 00:05:00 on ice.
5m 30s
Resuspend in 15 mL conical tube containing 2 mL warmed SOC media +100 micromolar (µM) DAP if using Jke201.
Note
Pre-warm SOC broth.
To make SOC broth:
Add close to 1 L MQ H2O and pH 7 with NaOH.
Incubate for 01:00:00 37 °C shaking .
1h
Streak out 50 µL , 100 µL , and 150 µL onto LB agar plates containing100 micromolar (µM) DAP if using Jke201 and 50 µg/mL Kanamycin.
Incubate at 37 °C Overnight .
Step 6: Confirm Inserts in Suicide Vector via PCR
6h 7m 40s
Label and culture colonies growing on plates in 5 mL LB with 50 micromolar (µM) Kanamycin (+ 100 micromolar (µM) DAP if using Jke201) Overnight at 37 °C shaking .
30s
Prep and store glycerol stocks of PCR'd colonies for future use.
Note
Pre-warm elution buffer in 37 °C incubator.
Prepare master mix using pFOK check primers and the recipe below:
Note
Use pFOK as control.
At thermocycler, select AO check, edit step #3 for Tm, #4 for extension time. pFOK Tm= 60.85 °C , extension time = 00:02:20
2m 20s
Run gel to confirm inserts are in suicide vector using recipe below:
To make TAE buffer dilute below recipe to 1X:
Add close to 1 L MQ H2O. For 1x combine 200 mL 50x with 9.8 L MQ H2O
Set up Owl EasyCast B2 Mini Gel Electrophoresis System tank ensuring orange rubber seal is against the wall and label is facing forward.
Microwave buffer and agarose for 00:00:30 , swirl to mix.
30s
Continue microwaving in 10-15 second increments until all agarose is dissolved.
Bring to point of boiling twice.
Once dissolved, add 2 µL SYBR Safe DNA Gel Stain and mix well.
Pour into mold, insert combs, and let sit for at least 01:00:00 but no more than 03:00:00 .
Note
Use 20 well comb, insert longer side (1.5).
4h
After gel is solidified, remove combs and rotate gel ensuring anode (red) is closest to you and wells are at the opposite end.
Fill tank to "Fill Line" with TAE buffer, and load samples.
Load5 µL DNA ladder in first well (5 kb).
Load 20 µL sample from PCR into wells, ensuring anode (red) is closest to you and wells are at the opposite end.
Turn on PowerPac Basic Power Supply to 120 V and run for approximately 01:00:00 .
Note
Once sample front is close to the end, stop run.
1h
Image Gel
Use Blot/UV/Stain-Free Tray. Clean with H2O before and after.
Take photo of gel and inspect colonies.
Expected result
There will be a single band at 1400 bp if colony has insert in suicide vector. pFOK control should show small band ~300 BP.
Grow cultures of colonies with confirmed insert in 5 mL LB with 50 micromolar (µM) Kanamycin (+ 100 micromolar (µM) DAP if using Jke201) Overnight at 37 °C shaking .
1h
After growing cultures overnight, prep out plasmid by repeating steps 40-41.
Select "plasmid" and "standard low concentration". pFOK is ~7 kb.
Note
Double recommended volume, for example, send 20 uL instead of 10 uL.
Prepare sample in PCR tube and label as instructed below:
Print out copy of order information and take to Plasmidsaurus drop box located on the 4th floor of the Couch Biomedical Research Building.
Step 7: Selection of the First Homologous Recombination Event (Conjugation)
22h 6m
Once sequence is confirmed, grow culture of E.coli containing confirmed plasmid and culture of desired recipient strain (e.g TOP52) in 2 mL LB broth (+ 100 micromolar (µM) DAP if using Jke201) overnight at 37 °C , shaking.
Note
Ensure you have autoclaved 22 mm filter membranes for next day
The next day, take 3 LB agar plates and put in incubator to pre-dry.
Place 500 µL from each culture (3 replicates) into sterile 1.5 mL tubes and spin down for 10.000 x g, 00:02:00
2m
Discard supernatant, resuspend in 500 µL , combine donor and recipient samples into 1 tube, spin down for 10.000 x g, 00:02:00 .
2m
Resuspend pellet in 50 µL LB and deposit on 22 mm filter membranes with 0.45 μm pores (Millipore, Merck) on pre-dried LB agar plate.
Mate for 06:00:00 at 37 °C .
6h
Label 1 50 mL conical for each plate. Use forceps to place filter from plate into conical, ensuring to put back of filter on white label part of the conical.
Wash filter using 1 mL LB, repeating until filter is clear.
Streak out 50 µL , 100 µL , and 150 µL (for each replicate) onto LB agar plates containing 50 µg/mL Kanamycin overnight at 37 °C , for at least 16:00:00 .
16h
Step 8: Counter-Selection of Second Homologous Recombination Event
5h 30m
Combine 6 colonies from each set of plates and combine into 4 mL LB (3 replicates). Grow for 04:00:00 at 37 °C , shaking.
4h
Make LB-no salt plates using the recipe below:
Note
Make Fresh Plates Every Time (Use Within < 1 Week).
On heated stir plate in 1 L glass with stir bar, add half of SMQ H2O and sucrose.
Turn on spin and heat up to 100-200°C, let sucrose dissolve before adding other ingredients.
Add remainder of SMQ H2O, Tryptone, yeast, and agar.
Autoclave solution.
Note
DO NOT ADD AHT UNTIL AFTER AUTOCLAVING.
After autoclaving, place in water bath at 50 °C and allow to cool for 01:00:00 .
1h
Add AHT, wrap in foil, and mix using magnetic stir plate.
Note
From now on, keep plates always protected from light.
Foil the agar bottle while you pour and cover the plates with foil while they solidify/cool.
Foil the bags or put them in a cabinet/box in the cold room.
Once thoroughly mixed, pour ~20 mL per plate, cover with foil, and let sit for at least 00:30:00 .
30m
Streak out 100 µL , 50 µL , and 25 µL onto fresh LB-no salt agar plates.
Incubate at 28 °C , protected from light, for at least 24 hours.
Step 9: Screen Colonies to Confirm Desired Mutation via PCR with External Primers
30s
Label 16-24 individual colonies on LB no salt plates.
Create master mix using outer check primers with recipe below:
Note
Use TOP52 gDNA for control. Should see band larger than 1400 BP.
Directly add colony scraped from plate to PCR tube containing 20 µL of master mix.
Repeat steps 41-43.2.
Grow cultures of colonies with confirmed insert in 5 mL LB Overnight at 37 °C shaking .
Note
Do not add abx or DAP
After growing cultures overnight, prep DNA by repeating steps 16-16.20.
Select "genome", "bacteria", and "standard".
Note
Double recommended volume, for example, send 40 uL instead of 20 uL.
Prepare sample in PCR tube and label as instructed below:
Print out copy of order information and take to Plasmidsaurus drop box located on the 4th floor of the Couch Biomedical Research Building.
Protocol references
Cianfanelli, F.R., Cunrath, O. & Bumann, D. Efficient dual-negative selection for bacterial genome editing. BMC Microbiol 20, 129 (2020). https://doi.org/10.1186/s12866-020-01819-2