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Method Article
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July 8th, 2025
Source: Martinez, E. et al., Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants. J. Vis. Exp. (2015)
This video demonstrates a method for assessing the intracellular growth of GFP-tagged Coxiella mutants within cultured epithelial cells. The mutant phenotype is visualized using immunostaining and epifluorescence microscopy, allowing the study of the effect of the mutation on intracellular growth.
1. Generation of a library of green fluorescence protein, GFP-tagged Coxiella transposon mutants
Manipulate Coxiella burnetii RSA439 NMII in a biosafety containment level 2 (BSL-2) in a microbial safety cabinet (MSC) in compliance with local rules. If compatible with the bacterial model used, repeat steps from 1.4.1 to 1.4.4 to increase the probability of obtaining clonal mutants. A typical mutant library is composed (at least) of a number of mutants that is equal to three times the number of coding sequences annotated in the genome of the organism used.
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Citric acid | Sigma | C0759-500G | ACCM-2 medium component |
| Sodium citrate | Sigma | S4641-500G | ACCM-2 medium component |
| Potassium phosphate | Sigma | 60218-100G | ACCM-2 medium component |
| Magnesium chloride | Sigma | M2670-100G | ACCM-2 medium component |
| Calcium chloride | Sigma | C5080-500G | ACCM-2 medium component |
| Iron sulfate | Sigma | F8633-250G | ACCM-2 medium component |
| Sodium chloride | Sigma | S9625-500G | ACCM-2 medium component |
| L-cysteine | Sigma | C6852-25G | ACCM-2 medium component |
| Bacto Neopeptone | BD (Beckton-Dickinson) | 211681 | ACCM-2 medium component |
| Casamino acids | BD (Beckton-Dickinson) | 223050 | ACCM-2 medium component |
| Methyl-B-Cyclodextrin | Sigma | C4555-10G | ACCM-2 medium component |
| RPMI w/glutamax | Gibco | 61870-010 | ACCM-2 medium component |
| RPMI w/glutamax, without phenol red | Gibco | 32404-014 | For cell culture and infection |
| Fetal Bovine Serum | GE healthcare | SH30071 | For cell culture |
| Trypsin EDTA (0.25%) | Life Technologies | 25200-056 | For cell culture |
| Saponin | Sigma | 47036 | For immunofluorescence staining |
| Ammonium chloride | Sigma | A9434 | For immunofluorescence staining |
| Bovine Serum Albumine | Sigma | A2153 | For immunofluorescence staining |
| Electroporation cuvette 0.1 cm | Eurogentec | ce0001-50 | For Coxiella transformation |
| Trackmates screw top tubes with caps | Thermo Scientific | 3741 | 2D barcoded screwcap |
| 96-well microplate with black walls and bottom | Greiner | 655076 | Flat dark bottom, for dsDNA quantitation |
| 96-well microplate, clear, black walls | Greiner | 655090 | Flat transparent bottom, for cell culture and imaging |
| 96-well PCR microplate | Biorad | 2239441 | DNAse/RNAse free |
| 96-well plate, Deepwell | Labcon | 949481 | For Coxiella mutants infections |
| PicoGreen | Life Technologies | P7581 | For dsDNA quantitation |
| Triton X-100 | Sigma | T9284-500ML | For dsDNA quantitation |
| Phusion High fidelity DNA Polymerase | New England Biolabs | M0530L | For single primer colony PCR |
| Celltracker Red CMTPX | Life Technologies | C34552 | For imaging |
| Anti-LAMP1 antibody | Sigma | 94403-1ML | For immunofluorescence staining |
| Hoechst 33258 | Sigma | L1418 | For imaging |
| Fluorescence microplate reader Infinite 200 Pro | Tecan | ||
| Epifluorescence automated microscope Cellomics | Thermo Scientific |
