Screening was carried out as described in the BACTH System Kit from Euromedex. Specific to our study, PCR amplification of wildtype and truncated imuA and imuB were amplified from protein expression vectors, before cloning into vectors pUT18, pUT18C, pKT25, and pKNT25 as described in the manufacturer's protocols (Euromedex), to fuse the appropriate Bordetella pertussis adenylate cyclase T18 or T25 fragments to the protein of interest. A complete list of plasmids created can be found in Table S8 . pUT18C::zip and pKT25::zip were used as positive controls and supplied in the BACTH System Kit (Euromedex). Combinations of vectors were cotransformed into E. coli BHT101 (Euromedex). Bacteria were grown in 50 μg/mL kanamycin, 100 μg/mL ampicillin, and 0.5 mM IPTG in LB broth overnight at 37°C. Two microliters of culture was spotted onto LB agar plates containing 50 μg/mL kanamycin, 100 μg/mL ampicillin, 0.1 or 0.5 mM IPTG, and 40 μg/mL 5‐bromo‐4‐chloro‐3‐indolyl‐β‐d‐galactopyranoside (X‐Gal). The plates were incubated for 24 h at 30°C, prior to imaging. Colonies with positive interactions between proteins of interest were identified by blue‐white screening. Here, the interaction of T18 and T25 fragments results in full‐length adenylate cyclase converting ATP to cAMP, and in turn, promotes the expression of β‐galactosidase, X‐gal hydrolysis, and the formation of blue‐pigmented colonies.
Bacth system kit
Manufactured by Euromedex
Sourced in France
The BACTH system kit is a laboratory equipment designed for the study of protein-protein interactions. The kit provides the necessary components to perform the Bacterial Adenylate Cyclase-based Two-Hybrid (BACTH) assay, which is a widely used technique for the detection and analysis of protein-protein interactions in bacterial cells.
Automatically generated - may contain errors
Lab products found in correlation
13 protocols using bacth system kit
1
Bacterial Adenylate Cyclase-Based Two-Hybrid Assay
2
Bacterial Two-Hybrid Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bacterial two-hybrid assays were performed using the BACTH System kit (Bacterial adenylate cyclase two-hybrid system kit, Euromedex) (Karimova et al., 1998 (link)). Dip was cloned into the high copy number vectors fused to the N-terminal (pUT18) and C-terminal (pUT18C) end of the T18 domain of adenylate cyclase and into the low copy number vectors fused to the N-terminal (pN-25) and C-terminal (pKT25) end of the T25 domain (Claessen et al., 2008 (link)). The components/fragments of the RNA degradosome (Figure 2A ) were cloned in pUT18C and pN-25. To screen for interactions each combination of phage and bacterial genes/fragments was co-transformed. Dilutions of an overnight culture were spotted on synthetic minimal M63 medium. β-galactosidase activity was measured quantitatively using a Miller assay (Zhang and Bremer, 1995 (link)).
3
Bacterial Two-Hybrid Analysis of MprF Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MprF-domain interactions were analyzed with a bacterial two-hybrid kit (BACTH system kit; Euromedex), as described recently (20 (link)). Briefly, E. coli BTH101 was transformed with mprF variants (Table S1 ) and fused to adenylate cyclase fragments T25 and T18 of Bordetella pertussis , and protein interactions resulting in cyclic AMP (cAMP) production and subsequent expression of the lac and mal operons in E. coli were quantified by determining β-galactosidase activity in triplicate (20 (link)).
4
Bacterial Adenylate Cyclase-Based Two-Hybrid
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmids used for the BACTH assay (Supplementary Table S1 ) were transformed into E. coli BTH101 (BACTH System Kit, Euromedex, Souffelweyersheim, France) [29 (link)]. Transformants were plated onto LB medium containing 100 μg/mL ampicillin and 50 μg/mL kanamycin, 200 μg/mL 5-bromo-4-chloro-3-indolyl-β-d -galactopyranoside (X-Gal), and 1 mM IPTG. Strains were induced with 1 mM IPTG for 3 h after dilution of OD600 to ~0.05, and OD600 was measured. Cultures were diluted 1:5 into PM2 (70 mM Na2HPO4·12H2O, 30 mM NaH2PO4·H2O, 1 mM MgSO4, and 0.2 mM MnSO4, pH 7.0) [45 (link)]. To permeabilize cells, 30 μL of toluene and 35 μL of a 0.1% SDS solution were added to 2.5 mL of bacterial suspension. The tubes were vortexed for 10 s and incubated at 37 °C for 45 min for evaporation of toluene. For the enzymatic reaction, 250 μL of permeabilized cells was added to PM2 supplemented with β-mercaptoethanol (final concentration, 100 mM) to a final volume of 1 mL. The reaction was started by adding 250 μL of 4 mg/ mL 1 O-nitrophenol-galactoside in PM2. Na2CO3 was added to stop the reaction and OD420 was measured [46 (link)]. The β-galactosidase activity, A (in units per milliliter), was calculated according to the following equation: A = 200 × ((OD420 of the culture − OD420 in the control tube)/minutes of incubation ×dilution factor)/ OD600.
5
Investigating PmlR2-PmSB-LOV Protein-Protein Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein–protein interaction between PmlR2 and PmSB-LOV was investigated with a BACTH system kit (EUROMEDEX, Souffelweyersheim, France)37 (link) according to the manufacturer’s instructions. The genes encoding PmlR2 and PmSB-LOV were PCR-amplified with primers R2T-F/R2T-R (Table S3 ) for pmlR2 and LOVT-F/LOVT-R (Table S3 ) for pmSB-LOV. The amplicons were digested with XbaI and KpnI, and then cloned into the same site of pUT18C and pKT25. PmlR2 and PmSB-LOV were fused at the C-terminus of the split adenylate cyclase domains (T18 and T25). The nucleotide sequence was confirmed by Eurofins Genomics. The pUT18C and pKT25 derivatives were co-transformed into a cya-deficient strain of E. coli BTH101, supplied by the manufacturer. β-galactosidase assays were performed as previously described38 (link).
6
Bacterial Two-Hybrid Interaction Studies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The plasmids used in bacterial two‐hybrid (BTH) analyses are listed in Supplementary Table 1. BTH interaction studies were performed as previously described (Karimova et al., 1998 ). The assays of β‐galactosidase activity in liquid cultures were performed as described in the BACTH System Kit, Euromedex. Details of the parA mutant library construction in the pKT25 plasmid and its screening for ParA proteins that failed to interact with DivIVA are provided in the Supporting Information file. The cloning of the M. smegmatisparA gene into the BTH plasmids allowed the production of protein starting with the sequence MDTP, which is different from the sequence annotation in databases but, according to our earlier observation, corresponds to the N‐terminus of the native protein. E. coli co‐localisation assays are described in the Supporting Information.
7
Two-Hybrid Assay for Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The two-hybrid assay was performed using the BACTH System Kit (Euromedex). Full-length cDNA for all six genes were amplified using primers with internal restriction sites (Table 2 ). After digestion of the PCR products, the inserts were ligated into linearized and dephosphorylated pKT25 and pUT18C vectors and used to transform E. coli. All ligations in this work were performed with the ReadyToGo ligation kit (GE Healthcare) and were transformed into NEB 10-β Competent E. coli cells (New England Biolabs), unless otherwise stated. Correct insertions were confirmed with vector specific primers (Table 2 ) followed by sequencing. Successful clones were co-transformed into electrocompetent BTH101 cells and selected on LA plates supplemented with ampicillin (100 μg/ml) and kanamycin (50 μg/ml). The protein-protein interactions were assayed according to the manufacturer’s protocol with the following modifications. One fresh colony of each interaction was transferred to 100 ml conical flasks with 5 ml LB supplemented with ampicillin 50 μg/ml, kanamycin 50 μg/ml and 0.5 mM IPTG, and incubated with shaking at 100 rpm at 20°C for 72 h. The extent of protein-protein interaction was measured with β-galactosidase assays as units/mg dry weight.
8
Cloning and Protein-Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GlmR and GlmS from both L. monocytogenes and B. subtilis were cloned in-frame into vectors pU18, pU18C, pKT25, and pKNT25 from the BACTH System kit (Euromedex) using XbaI and KpnI. Constructs were made originally in TAM1 or XL1-Blue E. coli and then moved to BTH101 E. coli for testing. Both blue/white screening on X-gal plates and β-galactosidase assays were carried out as previously described (32 (link)).
9
BACTH Assay for Detecting Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The BACTH Assay was carried out using the Euromedex BACTH System Kit. The target genes were first cloned into the BACTH plasmids (pKT25, pKNT25, pUT18 and pUT18C) in frame with either T25 or T18 fragments of the cyaA gene at the 5′ or 3′ end of the gene of interest (Table 2 ), to allow co-expression of fusion proteins. Then one of the T25 derived plasmid constructs was co-transformed with a T18 derived plasmid construct into E. coli BTH101 reporter cells and incubated on MacConkey/maltose agar [4% (w/v) MacConkey agar base (DifcoTM), 1% (w/v) maltose, 0.5mM IPTG and appropriate antibiotics) for 2 nights at 30°C.
When the two proteins of interest interacted with each other, heterodimerization of the fusion proteins allowed the complementation of the T25 and T18 fragments to form a catalytic domain of adenylate cyclase (CyaA), thus cAMP was synthesized. Then the mal operon was activated by cAMP/CAP complex. Therefore the maltose metabolism pathway was switched on in the E. coli BTH101 reporter strain, which allowed the fermentation of maltose and the production of acid that turned the pH indicator in MacConkey agar pink. Therefore positive interaction colonies were pink/red in color whereas negative colonies were white.
When the two proteins of interest interacted with each other, heterodimerization of the fusion proteins allowed the complementation of the T25 and T18 fragments to form a catalytic domain of adenylate cyclase (CyaA), thus cAMP was synthesized. Then the mal operon was activated by cAMP/CAP complex. Therefore the maltose metabolism pathway was switched on in the E. coli BTH101 reporter strain, which allowed the fermentation of maltose and the production of acid that turned the pH indicator in MacConkey agar pink. Therefore positive interaction colonies were pink/red in color whereas negative colonies were white.
10
BACTH Assay for Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BACTH assays were performed using the EUROMEDEX BACTH system kit. Expression constructs were transformed into JM109 E. coli cells to analyze protein synthesis. For this, bacterial cultures were induced with IPTG (isopropyl-β-D-thiogalactopyranoside; 2mM final concentration) at an OD600 of 0.6–0.8 and incubated on a rotary shaker for 2h at 37°C. Bacterial cells were collected by centrifugation, resuspended in Laemmli buffer, and analyzed by immunoblotting, using a FLAG epitope-specific antibody.
For protein-protein interaction studies, expression constructs encoding T18 and T25 fusion proteins were cotransformed into chemically competent DHM1 or BTH101 E. coli strains, and transformants were plated on LB plates containing kanamycin and gentamicin. Four colonies per transformation were used to inoculate LB overnight cultures with appropriate antibiotics, which were incubated overnight at 30°C on a rotary shaker. Two μl of the overnight cultures was spotted on selective LB plates containing X-gal (5-bromo-4-chloro-3-indolyl-β-d -galactopyranoside; 40μg/ml) and 2mM IPTG. The plates were incubated at 22°C, and the color of the colonies was monitored over a period of three to 5days. The experiments were performed at least three times with four different transformants from independent cotransformations.
For protein-protein interaction studies, expression constructs encoding T18 and T25 fusion proteins were cotransformed into chemically competent DHM1 or BTH101 E. coli strains, and transformants were plated on LB plates containing kanamycin and gentamicin. Four colonies per transformation were used to inoculate LB overnight cultures with appropriate antibiotics, which were incubated overnight at 30°C on a rotary shaker. Two μl of the overnight cultures was spotted on selective LB plates containing X-gal (5-bromo-4-chloro-3-indolyl-β-
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!