The Bacterial adenylate cyclase two-hybrid (BACTH) system kit from Euromedex was employed for conducting bacterial two-hybrid assays. Recombinant plasmids were obtained by amplifying the coding sequences of AbiEi and AbiEii and ligating them to pKT25 and pUT18C by restriction digestion, respectively. pKT25-AbiEi and pUT18C-AbiEii were co-transformed into the BTH101 strain, which serves as the reporter strain for the BACTH assay. X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 40 μg/ml) and IPTG (0.5 mM) were added to the positive clone medium and detected at 30°C for 24–72 hours. The plasmids pKT25-zip and pUT18C-zip were used as positive controls, while pKT25 and pUT18C served as negative controls. The experimental results were then recorded.
Bacterial adenylate cyclase two hybrid system kit
Manufactured by Euromedex
The Bacterial Adenylate Cyclase Two-Hybrid System Kit is a tool designed for the detection and analysis of protein-protein interactions in bacterial cells. The kit utilizes the principle of adenylate cyclase complementation to facilitate the identification of interacting proteins.
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10 protocols using bacterial adenylate cyclase two hybrid system kit
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Bacterial Two-Hybrid Assay for Protein-Protein Interactions
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Bacterial Two-Hybrid Analysis of RtcB, RtcA, and RtcR
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Bacterial 2-hybrid assays were performed using the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) System Kit (Euromedex) according to the manufacturer’s instructions. Full-length RtcB, RtcA and RtcR together with truncated forms of RtcR lacking one or two functional domains (RtcR1-186, RtcR1-353, RtcR187-353, RtcR187-532, RtcR354-532) were cloned in fusion with at either the N or the C-termini of the T18 fragment (vectors pUT18 and pUT18C) or of the T25 fragment (vectors pKT25 and pKNT25). The plasmids were co-transformed in pairs in the reporter strain for BACTH assay BTH101 and interactions between the hybrid proteins leading to lacZ reporter expression were monitored by β-galactosidase assays. Empty pUT18, pUT18C, pKT25 and pKNT25 vectors co-transformed in pairs were used as negative controls. Plasmids pKT25-zip and pUC18C-zip were used as positive controls. Plasmids pUT18C-HrpS, pUT18C-HrpV, pKT25-HrpS and pKT25-HrpV54 (link) were used to confirm the specificity of the observed interactions.
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Bacterial Two-Hybrid Assay for FHbp-SecA Interaction
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The protein–protein interaction of MC58 FHbp and L91543 FHbp with SecA was investigated using the Bacterial Adenylate Cyclase Two-Hybrid System Kit (Euromedex) according to manufacturer’s instructions. First, fHbp from MC58 and from L91543 was PCR-amplified with the primer pair BamHIfwd_MC58FHbp and EcoRIrev_MC58FHbp and primer pair BamHIfwd_L91543FHbp and EcoRIrev_L91543FHbp respectively (Table 3 ) then the PCR products cloned separately into vector pUT18. The gene encoding SecA from MC58 was PCR-amplified with primer pair, PstIfwd_SecA and SmaIreverse_SecA (Table 3 ) then cloned into vector pKT25. 25–50 ng of the appropriate prey (pKT25-based construct) and the equivalent concentration of appropriate bait (pUT18-based construct) were co-transformed into 100 μl of competent E. coli BTH101 cells and plated on MacConkey agar containing 0.5 mM IPTG and appropriate antibiotics. Bacteria expressing interacting hybrid proteins formed pink/purple colonies while cells expressing non-interacting proteins remained white/light pink. As a positive control, a co-transformant containing commercial pKT25-zip and pUT18-zip constructs was used. Co-transformants containing empty vector pKT25 and/or pUT18 were used as negative controls. Pink colonies were isolated and grown in LB broth and plasmid DNA extracted for verification by PCR and sequencing.
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Bacterial Adenylate Cyclase Two-Hybrid Assay
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To detect protein–protein interactions among the RBP enzymes, the bacterial adenylate cyclase two-hybrid system (18 (link)) was used as described previously (27 (link)). Briefly, the coding regions of S. meliloti RF biosynthetic enzymes and SMc02977 and B. abortus bab2_0247 were PCR amplified and cloned into the BamHI/KpnI restriction sites of pUT18C and pKT25 plasmids. The constructs were verified by PCR and sequencing. The cya-deficient E. coli strain DHM1 was cotransformed with the pUT18C and pKT25 derivatives. Transformants were selected on LB plates supplemented with kanamycin (40 μg/ml), ampicillin (100 μg/ml), and nalidixic acid (15 μg/ml). A positive interaction between two tested proteins produced a functional adenylate cyclase leading to cAMP synthesis and lacZ expression. The β-galactosidase activities were determined according to the procedure by Bacterial Adenylate Cyclase Two-Hybrid System kit (Euromedex; catalog no.: EUK001). Measurements of β-galactosidase activity were made in triplicate, and the experiments were repeated at least twice.
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Bacterial Adenylate Cyclase Two-Hybrid Assay
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Bacterial Two-Hybrid Crotonylation Assay
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Bacterial two hybrids for protein–protein interaction were performed with the Bacterial Adenylate Cyclase Two-Hybrid System Kit (Euromedex) according to the product manual. For in vitro crotonylation reaction, 1 µg of Kct1, 2 µg of Glk, and 10 mM of crotonyl-CoA (Sigma) were added in the reaction buffer (20 mM Tris-HCI (pH 8.0), 100 mM KCl, 7.5 mM MgCl2) and incubated at 30 °C for 1.5 h, followed by Glk activity assays or loading for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Decrotonylation assays were performed in the same reaction conditions, but 2.5 µg of Glk + 2 µg of CobB were used. The total loading protein and crotonylation were analyzed by western blot with antibodies against His-tag (Sigma) and crotonyllysine (Kcr) (PTM-502, PTM Biolabs Inc.) (1:2000), respectively.
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Bacterial Adenylate Cyclase Two-Hybrid System
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To investigate interaction between the permease IsdF and the ATPases IsdL and FhuC, the commercially available bacterial adenylate cyclase two-hybrid system kit was used (Euromedex). In brief, E. coli BTH101 was co-transformed with pKT25:isdF and pUT18C:isdL or pUT18C:fhuC, respectively. In case of protein–protein interaction, the catalytic domains T25 and T18 of the Bordetella pertussis adenylate cyclase are able to heterodimerize and to produce cyclic AMP allowing the expression of lacZ. This leads to blue colony formation on LB agar indicator plates containing 40 μg/ml X-Gal (Sigma-Aldrich/Merck), 0.5 mM IPTG (Thermo Scientific), 100 μg/ml ampicillin, and 50 μg/ml kanamycin after incubation for 2 days at 30 °C. As positive control, pKT25:zip and pUT18C:zip were used encoding a leucine zipper; as negative control, empty vectors were co-transformed into BTH101.
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Bacterial Two-Hybrid Assay for HD Protein Interactions
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In order to ascertain the possible interaction between HD1 and HD2 protein bacterial two-hybrid assays were performed using the Bacterial adenylate cyclase two-hybrid system kit from Euromedex. The cDNA of each of the HD genes (S1 File ) was synthesized at Eurofins and delivered as an insert in vector (pEX-K4). Synthetic HD genes were amplified by PCR (S11 Table ) and sub-cloned into plasmids pKNT25 and pUT18 using the Hind III and Pst I restriction sites present in the MCS of both of plasmids, according to standard molecular biology techniques and the Euromedex manual [52 (link)]. Genes were cloned in frame at the N-terminus end of the T25 and T18 peptides. After transformation, integrity of the constructs was assessed by PCR amplification and sequencing of the cloned fragments (primers MP191/MP192 and MP193/MP194) (S11 Table ). Different combinations of the recombinant plasmids (S4C Fig ) were co-transformed into BTH101 E. coli cells (S6 Table ). After successful transformation, the phenotype of 8 clones (identified as 1.1–10.8) of each of the different co-transformations was assessed in X-gal and MacConkey/maltose media upon incubation at 30°C for 48h. Results were scored after 24h and 48h incubation times. The presence of both fragments and correct plasmids in each of the clones was assessed by PCR (S11 Table ).
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Validating Novel Gene Interactions Using Bacterial Two-Hybrid Assay
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A total 14 pairs of predicted two-gene interactions and eight groups of predicted three-gene interactions and a pair of positive controls were tested (see Fig. S3). Among these interactions, six pairs of novel interactions between two genes modules and ten pairs of novel interactions among three gene modules, which were not predicted in the STRING database, were selected for experimental validation.
Strains and plasmids used in this study are listed in Table S6 in the supplemental material.S. suis strains were grown in tryptic soy broth supplemented with 10% bovine serum at 37°C under vigorous agitation. Escherichia coli BTH101strain was grown aerobically in lysogeny broth at 37°C. Bacterial two-hybrid analyses, including a MacConkey-maltose indicator plate assay and β-galactosidase assays, were performed as described in the manual of the bacterial adenylate cyclase two-hybrid system kit (Euromedex).
Each interaction pair was scored on MacConkey-maltose indicator plate assays on a minimum of three individual occasions, and β-galactosidase assays were performed at least three times.
Strains and plasmids used in this study are listed in Table S6 in the supplemental material.
Each interaction pair was scored on MacConkey-maltose indicator plate assays on a minimum of three individual occasions, and β-galactosidase assays were performed at least three times.
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Bacterial Adenylate Cyclase Two-Hybrid System
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The Bacterial Adenylate Cyclase Two‐Hybrid System Kit (Euromedex) was used according to manufacturer’s instructions. Briefly, plasmids pUT18C and pKT25 were used to construct N‐terminal fusions with T18 and T25 fragments of adenylate cyclase for spoIIDMP, spoIIQ and spoIIIAH. Combinations of pUT18C and pKT25‐based plasmids were transformed into chemically competent E. coli BTH101 cells which were plated onto LB agar supplemented with carbenicillin (100 μg/ml), kanamycin (50 μg/ml), X‐Gal (50 μg/ml) and 0.5 mM IPTG and grown at 30°C for 24 h to allow for complementation. pUT18C‐zip and pKT25‐zip plasmids encoding the GCN4 leucine zipper fused to T18 and T25 fragments of adenylate cyclase, respectively, were used together as positive controls and in combination with other plasmids as negative controls. Experiments were carried out in duplicate.
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