Dec 01, 2025
Transformation of Tn7 insertion elements across strains of Vibrio fischeri
Peer-reviewed method
- Andrew G. Cecere1,2,
- Chris A. Muriel-Mundo1,2,
- Derek J. Fisher3,4,
- Tim I. Miyashiro1,2,5
- 1Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA, USA;
- 2The One Health Microbiome Center, Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, USA;
- 3Multidisciplinary Biomedical & Biological Sciences, Southern Illinois University Carbondale, Carbondale, IL, USA;
- 4School of Biological Sciences, Southern Illinois University Carbondale, Carbondale, IL, USA;
- 5Correspondence: [email protected]
- PLOS ONE Lab ProtocolsTech. support email: [email protected]
External link: https://doi.org/10.1371/journal.pone.0338632
Protocol Citation: Andrew G. Cecere, Chris A. Muriel-Mundo, Derek J. Fisher, Tim I. Miyashiro 2025. Transformation of Tn7 insertion elements across strains of Vibrio fischeri. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg31k11l25/v1
Manuscript citation:
Cecere AG, Muriel-Mundo C, Fisher DJ, Miyashiro TI (2025) Transformation of Tn7 insertion elements across strains of Vibrio fischeri. PLOS One 20(12). doi: 10.1371/journal.pone.0338632
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: September 08, 2025
Last Modified: December 01, 2025
Protocol Integer ID: 226768
Keywords: strains of vibrio fischeri, genetic content at the tn7 insertion, transformation of tn7 insertion element, tn7 insertion, tn7 insertion element, vibrio fischeri, strain, strain es114, dependent natural transformation, genetic content
Funders Acknowledgements:
National Institute of General Medical Sciences
Grant ID: R35 GM152259
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Abstract
This protocol details how to use pLosTfoX-dependent natural transformation to transform non-canonical strains of Vibrio fischeri with genetic content at the Tn7 insertion of strain ES114.
Materials
Materials needed:
1. Culture tubes, e.g., 14-mL Round-Bottom Polystyrene Test Tubes (Falcon)
2. Defined minimal medium (DMM) with N-acetylglucosamine (GlcNAc) – [50 mM MgSO4, 10 mM CaCl2, 300 mM NaCl, 10 mM KCl, 0.01 mM FeSO4, 0.33 mM K2HPO4, 50 mM Tris-HCl (pH 7.5), 0.2% GlcNAc]. Store at 4°C, use within 24 hours.
3. Chloramphenicol (cam)
4. 125-mL baffled Erlenmeyer flask
5. LBS medium [1% (w/v) tryptone, 0.5% (w/v) yeast extract, 2% (w/v) NaCl, 50 mM Tris–HCl (pH 7.5)], with 1.5% w/v agar for solid medium
6. Shaking incubator at 28°C (New Brunswick)
7. Spectrophotometer and cuvettes (Eppendorf)
8. Genomic DNA (extracted according to manufacturer's instructions, Biosearch Technologies Inc.)
9. LBS + 5.0 µg/mL erythromycin (erm) plates
Troubleshooting
Preparation of Vibrio fischeri Cultures
Initiate a starter culture by inoculating a Falcon tube containing 2 mL DMM-GlcNAc + 2.5 µg/mL cam with an isolated colony of a V. fischeri strain harboring pLosTfoX. Incubate starter culture overnight (~16 h) at 28°C shaking at 200 rpm.
Transfer 20 mL DMM + 2.5 µg/mL cam into a 125-mL baffled Erlenmeyer flask and incubate at 28°C to prewarm media.
Measure the OD600 of the starter culture. (Note: typically OD600 = 1.0–1.5)
Inoculate flask with 1 mL starter culture. Incubate at 28°C shaking at 200 rpm.
Preparation of natural transformation reactions
When the turbidity of culture is OD600 = 0.25, transfer 0.5 mL of the culture to two Falcon tubes. To one tube (experimental), add 0.5 µg genomic DNA substrate. To the other tube (no DNA control), add equivalent volume of vehicle. Gently vortex both tubes and incubate at room temperature statically overnight.
Selection of transformants
Add 0.5 mL LBS to each culture tube. Incubate shaking at 28°C.
After 90 minutes, plate 0.2 mL onto LBS-erm. To assess total viable cell count, perform 10-fold serial dilutions to 10-7 using 10-µL volumes into 90 µL LBS. Spot 10-µL volumes of 10-2–10-7 onto LBS plate with pre-drawn grids. Incubate plates at 28°C overnight.
For each sample, calculate the concentration of viable cells and transformants per mL. Calculate the transformation efficiency by dividing concentration of transformants by the concentration of DNA.
Streak purify ermR colonies onto LBS-erm for further isolation.