Computational protocol: Characterising resuscitation promoting factor fluorescent-fusions in mycobacteria

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Protocol publication

[…] For western blots of soluble extracts and precipitated fraction of disrupted cells, 10 ml cultures of the strains were grown in Hartmans-de Bont broth with the inducer tetracycline up to an OD600 of 1. The cultures were then centrifuged and resuspended in 500 μl PBS+ protease inhibitor (cOmplete, Sigma) and disrupted in a water bath sonicator. The soluble extract and the precipitated fraction were subsequently separated by centrifugation and protein concentration in the soluble extract was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher). An equal amount of soluble proteins for all the samples was loaded in the gels for western blotting. The precipitated fractions were resuspended in the same volume for all the samples and a fixed volume was loaded on the gels. Western blots were performed using the NuPAGE Western blotting system (Thermo Fisher). SeeBlue Pre-stained Protein Standard (Thermo Fisher) was used as size standard. As a primary antibody, polyclonal rabbit anti-EGFP antibody (Thermo Fisher, dilution 1:1000) or polyclonal rabbit anti-mCherry antibody (Novus Biologicals, dilution 1:1000) were used, and as a secondary antibody we used goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, dilution 1:5000). The blots were developed using the SuperSignal West Femto Substrate Trial Kit (Thermo Fisher) in a LAS-3000 Fuji Imager.For the western blots of filtrated culture supernatants, 100 ml of each strain were grown in Hartmans-de Bont broth without tween 80 and with the inducer tetracycline up to an OD600 of approximately 0.6. Protease inhibitor was added to the cultures 30 min before collecting, centrifuging and filtering the supernatants (0.22 μm). The proteins in the supernatants were concentrated by Ion-Exchange chromatography, adapting a published protocol []. The pI of the Rpfs-mCherry and Rpfs-EGFP fusion proteins was theoretically calculated using the Compute pI/Mw tool on the ExPASy Server []. It was found in all cases to be below the pH of the buffers (7.5) so the anion exchanger DEAE-Sepharose was used. 2 ml columns were washed with 5× volumes of water and then equilibrated with 5× volumes of buffer A (20 mM TrisHCl, pH 7.5; 20 mM KCl, 1 mM EDTA, 1 mM DTT). The supernatants were passed through the column and eluted with 3× volumes of buffer B (buffer A with 1 M NaCl). 2 ml samples of the first eluate were precipitated with 20% TCA and washed twice with acetone, dried and resuspended in loading buffer for its use in western blot, which was performed as previously explained.The theoretical molecular weight of the fusion proteins was calculated using the Compute pI/Mw tool on the ExPASy Server []. [...] M. smegmatis strains harbouring each Rpf-mCherry or m-Cherry alone were grown in shaking up to mid-log phase (OD600 ≈ 0.6) in Sauton’s medium before being exposed to different stress conditions. For osmotic stress, a NaCl solution in water was added to the cultures at a 1 M final concentration. For acidic stress, cultures were washed in pH 4.5 Sauton and resuspended in the same medium. For nutrient starvation, cultures were washed twice in PBS-0.025% Tyloxapol (Sigma) and resuspended in the same solution. Samples for CFU counts were taken at this point and subsequently, the cultures were incubated for 6 days at 37 °C in static. After the incubation, the cultures were washed and resuspended in Sauton’s medium, and samples were taken for CFU and MPN counts and resuscitation index calculations.Assessment of CFU counts was performed in serial dilutions of cultures in triplicate by using the standard droplet method [] and subsequent incubation of the agar plates by 48 h.For MPN counts, serial dilutions of 200 μl cultures samples were incubated in 96-well plates in Sauton’s medium containing the inducer tetracycline. Three wells were used for each dilution. The plates where incubated in static at 37 °C for 2 weeks and MPN calculated []. Three independent experiments were performed for each condition and strain. The potential for resuscitation of non platable cells of the different strains was expressed as the resuscitation index (RI): log10(MPN)-log10(CFU) []. Statistical differences in RI between strains were calculated using One Way Analysis of Variance (one-way ANOVA). We subsequently applied Bonferroni t-test, an all pairwise multiple comparisons procedure, to isolate the groups that differ from others. One-way ANOVA and Bonferroni t-test were applied using SigmaPlot (Systat Software, San Jose, CA). […]

Pipeline specifications

Software tools Compute pI/Mw tool, SigmaPlot
Databases ExPASy
Applications Miscellaneous, Protein physicochemical analysis
Organisms Corynebacteriales
Diseases Tuberculosis