N. gonorrhoeae strains FA1090 (ATCC 700825), MS11 (ATCC BAA1833), N400 [37 (link)] and N. meningitidis strains FAM20 [38 (link)], C311, C480, C462 [39 (link)] and JB515 [38 (link)] were grown at 37°C in 5% CO2 on GC agar plates (GC medium base; Acumedia, Neogen Corporation, Lansing, MI, USA). The PilE sequence variants 3:1, 5:1, 6:1, and 8:1 of MS11 [40 (link),30 (link)] and the PilT-deficient mutant of FAM20 [41 (link)] were described previously. E. coli strain DH5α was used for cloning and plasmid propagation, and strain BL21 (DE3) was used for protein expression. E. coli strains were maintained on LB agar plates (Acumedia). Liquid cultures of Neisseria were propagated in GC broth supplemented with Kellogg’s supplement. Liquid cultures of E. coli were grown in LB broth (Acumedia). Antibiotics were used at the following concentrations: 100 μg/ml for ampicillin and 50–100 μg/ml for kanamycin.
Lb broth
Manufactured by Acumedia
Sourced in United States
LB broth is a widely used nutrient-rich growth medium for culturing bacteria. It provides essential nutrients, including amino acids, vitamins, and minerals, to support the growth and proliferation of a variety of bacterial species.
Automatically generated - may contain errors
5 protocols using lb broth
1
Cultivation and Genetic Manipulation of Neisseria
2
Bacterial growth conditions and human cell line
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neisseria meningitidis serogroup C strain FAM20 and its pilE mutant (ΔpilE) have been previously described (Rahman et al., 1997 (link); Jones et al., 2009 (link); Engman et al., 2016 (link)). The strains were grown on GC agar (Acumedia) with 1% Kellogg’s supplements (Kellogg et al., 1968 (link)) at 37°C and 5% CO2 for 16-18 h before experiments. Lactobacillus reuteri ATCC PTA5289 (de Klerk et al., 2016 (link)) was grown at 37°C 5% CO2 on Rogosa (Oxoid) agar for 30-72 h and then in MRS broth for 16-18 h. Escherichia coli DH5α was grown on LA-plates and then in LB broth (Acumedia) at 37°C 5% CO2 at 200 rpm shaking.
The human pharyngeal epithelial cell line FaDu (ATCC-HBT43) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, GibcoTM) and supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich) at 37°C and 5% CO2 in a humidified environment.
The human pharyngeal epithelial cell line FaDu (ATCC-HBT43) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, GibcoTM) and supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich) at 37°C and 5% CO2 in a humidified environment.
3
Culturing A. actinomycetemcomitans and E. coli Strains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Aggregatibacter actinomycetemcomitans ATCC 700685 (strain HK1651, serotype b), ATCC 33384 (strain NCTC 9710, serotype c), ATCC 43717 (strain SUNYab 75, serotype a), ATCC 43719 (strain SUNYab 67, serotype c) were obtained from the American Type Culture Collection (Manassas). All A. actinomycetemcomitans strains used in this study were chloramphenicol and kanamycin sensitive. Bacteria were cultured in brain heart infusion (BHI) broth (Acumedia) and incubated at 37°C in an atmosphere supplemented with 5% CO2. E. coli 1354 (Barrett et al., 2008 (link)), a diaminopimelic acid (DAP) auxotroph, was cultured in LB broth (Acumedia) supplemented with 100 μg/mL DAP (Sigma) and incubated with aeration at 37°C.
4
Broth Microdilution Antimicrobial Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The broth microdilution process was employed with 96-well plates (TPP, Switzerland). Each extract was diluted twofold in LB broth® (Acumedia, Michigan, USA), and the wells were injected with 1 × 106 CFU of bacteria (in a 0.2 mL final volume). After incubation at 37 °C for 24 h, the MIC analysis was implemented consistent with the references of the Clinical and Laboratory Standards Institute54 . The extent of concentrations investigated for each suspension ranged from 0.062 to 64 µg/mL. The MIC value is defined as the lowest antimicrobial concentration that impedes the development of microorganisms, while the SIC value is defined as the antimicrobial concentration less than one capable of impeding the observed development and reproduction of the microorganism.
5
Construction and Maintenance of Recombinant Bacterial Strains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The plasmid and bacterial strains used are listed in Table 1 . The pValac plasmid was constructed by our group in 2009 (Guimarães et al., 2009 (link)). The L. lactis FnBPA+ lineage was developed by Que et al. (2001 (link)), who kindly gave it to the group of Dr. Philippe Langella from INRAe—France. Dr. Philippe Langella passed it on to us and, currently, both the plasmid and the lineage are part of our microbiological collection.
Escherichia coli TG1 was grown in LB broth (Acumedia, San Bernardino, United States) at 37°C with shaking. Lactococcus lactis FnBPA+ was grown at 30°C without shaking in M17 broth (Sigma-Aldrich, Darmstadt, Germany) enriched with 0.5% glucose (GM17). Recombinant bacteria were selected by addition of the following antibiotics: for L. lactis FnBPA+, erythromycin at 5 μg/ml; for L. lactis FnBPA+ (pValac:e6ag85a), erythromycin at 5 μg/ml and chloramphenicol at 10 μg/ml; and for E. coli (pValac:e6ag85a), chloramphenicol at 10 μg/ml.
Plasmids from E. coli and L. lactis were isolated as described by Green and Sambrook (2012 ), with the following modification: for L. lactis, TE-LYS (25% sucrose, 1 mmol EDTA, 50 mmol Tris–HCl, pH 8, lysozyme 10 mg/ml) was added to the samples for 1 h at 37°C before the addition of lysis solution.
Escherichia coli TG1 was grown in LB broth (Acumedia, San Bernardino, United States) at 37°C with shaking. Lactococcus lactis FnBPA+ was grown at 30°C without shaking in M17 broth (Sigma-Aldrich, Darmstadt, Germany) enriched with 0.5% glucose (GM17). Recombinant bacteria were selected by addition of the following antibiotics: for L. lactis FnBPA+, erythromycin at 5 μg/ml; for L. lactis FnBPA+ (pValac:e6ag85a), erythromycin at 5 μg/ml and chloramphenicol at 10 μg/ml; and for E. coli (pValac:e6ag85a), chloramphenicol at 10 μg/ml.
Plasmids from E. coli and L. lactis were isolated as described by Green and Sambrook (2012 ), with the following modification: for L. lactis, TE-LYS (25% sucrose, 1 mmol EDTA, 50 mmol Tris–HCl, pH 8, lysozyme 10 mg/ml) was added to the samples for 1 h at 37°C before the addition of lysis solution.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!