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In This Article

  • Overview
  • Protocol
  • Disclosures
  • Materials

Overview

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.

Protocol

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.

  1. Preparation of elect....

Disclosures

No conflicts of interest declared.

Materials

NameCompanyCatalog NumberComments
Citric acidSigmaC0759-500GACCM-2 medium component
Sodium citrateSigmaS4641-500GACCM-2 medium component
Potassium phosphateSigma60218-100GACCM-2 medium component
Magnesium chlorideSigmaM2670-100GACCM-2 medium component
Calcium chlorideSigmaC5080-500GACCM-2 medium component
Iron sulfateSigmaF8633-250GACCM-2 medium component
Sodium chlorideSigmaS9625-500GACCM-2 medium component
L-cysteineSigmaC6852-25GACCM-2 medium component
Bacto NeopeptoneBD (Beckton-Dickinson)211681ACCM-2 medium component
Casamino acidsBD (Beckton-Dickinson)223050ACCM-2 medium component
Methyl-B-CyclodextrinSigmaC4555-10GACCM-2 medium component
RPMI w/glutamaxGibco61870-010ACCM-2 medium component
RPMI w/glutamax, without phenol redGibco32404-014For cell culture and infection
Fetal Bovine SerumGE healthcareSH30071For cell culture
Trypsin EDTA (0.25%)Life Technologies25200-056For cell culture
SaponinSigma47036For immunofluorescence staining
Ammonium chlorideSigmaA9434For immunofluorescence staining
Bovine Serum AlbumineSigmaA2153For immunofluorescence staining
Electroporation cuvette 0.1 cmEurogentecce0001-50For Coxiella transformation
Trackmates screw top tubes with caps Thermo Scientific37412D barcoded screwcap
96-well microplate with black walls and bottom Greiner655076Flat dark bottom, for dsDNA quantitation
96-well microplate, clear, black wallsGreiner655090Flat transparent bottom, for cell culture and imaging
96-well PCR microplateBiorad2239441DNAse/RNAse free
96-well plate, DeepwellLabcon949481For Coxiella mutants infections
PicoGreenLife TechnologiesP7581For dsDNA quantitation
Triton X-100 SigmaT9284-500MLFor dsDNA quantitation
Phusion High fidelity DNA PolymeraseNew England BiolabsM0530LFor single primer colony PCR
Celltracker Red CMTPXLife TechnologiesC34552For imaging
Anti-LAMP1 antibodySigma94403-1MLFor immunofluorescence staining
Hoechst 33258SigmaL1418For imaging
Fluorescence microplate reader Infinite 200 ProTecan
Epifluorescence automated microscope CellomicsThermo Scientific

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