Structured Review

Thermo Fisher dh5α
Promoter activity in native and genetically engineered nlpD1 - penA intergenic regions. ( a ) Design of reporter lacZ reporter gene constructs. The indicated 238 bp DNA fragments shown by the thick lines (plus flanking SpeI and HindIII restriction sites) were obtained from PCR fragments amplified from 1026b and Bp1651 genomic DNA. The indicated −41C, −78G and −171T nucleotides present on the 1026b fragment were individually changed to −41T, −78A and −171C present in Bp1651. Lastly, a 532 bp DNA fragment (plus flanking SpeI and HindIII restriction sites) containing a 336 bp instead of a 42 bp nlpD1 terminus was obtained from a PCR fragment amplified from 1026b genomic DNA. These fragments were cloned into a mini-Tn 7 - lacZ transcriptional fusion vector and integrated into the Bp82.27 chromosome. ( b ) β-Gal activity in strains harboring the single-copy lacZ reporter constructs. The strains resulting from chromosomal integration were Bp82.363 (empty vector), Bp82.365 (1026b fragment), Bp82.365 (Bp1651 fragment), Bp82.366 (1026b fragment with −41T), Bp82.367 (1026b fragment with −78A), Bp82.371 (1026b fragment with −171C) and Bp82.372 (1026b fragment with a 331 bp instead of 42 bp nlpD1 3′ terminus. β-Gal activities were measured in triplicate on three separate days and are expressed in Miller units. Error bars indicate standard deviation from the mean. ( c ) The P A promoter is active in E . coli . A plasmid was constructed that contains a penA ’-‘ lacZ translational fusion under transcriptional and translation control of the 174 bp 1026b IR with the -78 G to A transition. The resulting pPZ10 penA ’-‘ lacZ thus contains the P A promoter. The first 7 amino acids of PenA are fused in-frame to LacZ. The resulting pPZ10 penA ’-‘ lacZ and the empty pPZ10 vector were transformed into strain <t>DH5α.</t> β-Gal activity was assessed by plating on an LB plate with X-Gal indicator (left) or by measuring hydrolysis of ortho-nitrophenyl-β-D-galactopyranoside (ONPG) in culture grown cells (right). The ONPG β-Gal assays were performed in triplicate and activity is expressed in Miller units. Error bars indicate standard deviation from the mean. The plate picture is a minimally processed section of an image of an agar plate shown in Fig. S6 .
Dh5α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Transcriptional and post-transcriptional regulation of PenA β-lactamase in acquired Burkholderia pseudomallei β-lactam resistance"

Article Title: Transcriptional and post-transcriptional regulation of PenA β-lactamase in acquired Burkholderia pseudomallei β-lactam resistance

Journal: Scientific Reports

doi: 10.1038/s41598-018-28843-7

Promoter activity in native and genetically engineered nlpD1 - penA intergenic regions. ( a ) Design of reporter lacZ reporter gene constructs. The indicated 238 bp DNA fragments shown by the thick lines (plus flanking SpeI and HindIII restriction sites) were obtained from PCR fragments amplified from 1026b and Bp1651 genomic DNA. The indicated −41C, −78G and −171T nucleotides present on the 1026b fragment were individually changed to −41T, −78A and −171C present in Bp1651. Lastly, a 532 bp DNA fragment (plus flanking SpeI and HindIII restriction sites) containing a 336 bp instead of a 42 bp nlpD1 terminus was obtained from a PCR fragment amplified from 1026b genomic DNA. These fragments were cloned into a mini-Tn 7 - lacZ transcriptional fusion vector and integrated into the Bp82.27 chromosome. ( b ) β-Gal activity in strains harboring the single-copy lacZ reporter constructs. The strains resulting from chromosomal integration were Bp82.363 (empty vector), Bp82.365 (1026b fragment), Bp82.365 (Bp1651 fragment), Bp82.366 (1026b fragment with −41T), Bp82.367 (1026b fragment with −78A), Bp82.371 (1026b fragment with −171C) and Bp82.372 (1026b fragment with a 331 bp instead of 42 bp nlpD1 3′ terminus. β-Gal activities were measured in triplicate on three separate days and are expressed in Miller units. Error bars indicate standard deviation from the mean. ( c ) The P A promoter is active in E . coli . A plasmid was constructed that contains a penA ’-‘ lacZ translational fusion under transcriptional and translation control of the 174 bp 1026b IR with the -78 G to A transition. The resulting pPZ10 penA ’-‘ lacZ thus contains the P A promoter. The first 7 amino acids of PenA are fused in-frame to LacZ. The resulting pPZ10 penA ’-‘ lacZ and the empty pPZ10 vector were transformed into strain DH5α. β-Gal activity was assessed by plating on an LB plate with X-Gal indicator (left) or by measuring hydrolysis of ortho-nitrophenyl-β-D-galactopyranoside (ONPG) in culture grown cells (right). The ONPG β-Gal assays were performed in triplicate and activity is expressed in Miller units. Error bars indicate standard deviation from the mean. The plate picture is a minimally processed section of an image of an agar plate shown in Fig. S6 .
Figure Legend Snippet: Promoter activity in native and genetically engineered nlpD1 - penA intergenic regions. ( a ) Design of reporter lacZ reporter gene constructs. The indicated 238 bp DNA fragments shown by the thick lines (plus flanking SpeI and HindIII restriction sites) were obtained from PCR fragments amplified from 1026b and Bp1651 genomic DNA. The indicated −41C, −78G and −171T nucleotides present on the 1026b fragment were individually changed to −41T, −78A and −171C present in Bp1651. Lastly, a 532 bp DNA fragment (plus flanking SpeI and HindIII restriction sites) containing a 336 bp instead of a 42 bp nlpD1 terminus was obtained from a PCR fragment amplified from 1026b genomic DNA. These fragments were cloned into a mini-Tn 7 - lacZ transcriptional fusion vector and integrated into the Bp82.27 chromosome. ( b ) β-Gal activity in strains harboring the single-copy lacZ reporter constructs. The strains resulting from chromosomal integration were Bp82.363 (empty vector), Bp82.365 (1026b fragment), Bp82.365 (Bp1651 fragment), Bp82.366 (1026b fragment with −41T), Bp82.367 (1026b fragment with −78A), Bp82.371 (1026b fragment with −171C) and Bp82.372 (1026b fragment with a 331 bp instead of 42 bp nlpD1 3′ terminus. β-Gal activities were measured in triplicate on three separate days and are expressed in Miller units. Error bars indicate standard deviation from the mean. ( c ) The P A promoter is active in E . coli . A plasmid was constructed that contains a penA ’-‘ lacZ translational fusion under transcriptional and translation control of the 174 bp 1026b IR with the -78 G to A transition. The resulting pPZ10 penA ’-‘ lacZ thus contains the P A promoter. The first 7 amino acids of PenA are fused in-frame to LacZ. The resulting pPZ10 penA ’-‘ lacZ and the empty pPZ10 vector were transformed into strain DH5α. β-Gal activity was assessed by plating on an LB plate with X-Gal indicator (left) or by measuring hydrolysis of ortho-nitrophenyl-β-D-galactopyranoside (ONPG) in culture grown cells (right). The ONPG β-Gal assays were performed in triplicate and activity is expressed in Miller units. Error bars indicate standard deviation from the mean. The plate picture is a minimally processed section of an image of an agar plate shown in Fig. S6 .

Techniques Used: Activity Assay, Construct, Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation, Standard Deviation, Transformation Assay

Related Articles

Clone Assay:

Article Title: Genetic identification of potential RNA-binding regions in a group II intron-encoded reverse transcriptase
Article Snippet: .. BL21(DE3) and BL21(DE3)-R3/Rosetta (Novagen) were used for LtrA protein expression , and DH5α, DH10B, and OmniMAX 2-T1R cells (Invitrogen) were used for plasmid DNA preparation and cloning. ..

Article Title: Identification and Characterization of a New Enoyl Coenzyme A Hydratase Involved in Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates in Recombinant Escherichia coli
Article Snippet: .. E. coli XL1-Blue (Stratagene Cloning Systems, La Jolla, Calif.) and DH5α (Invitrogen Life Technologies, Carlsbad, Calif.) were used as host strains for general cloning works and gene expression. ..

other:

Article Title: Error-Prone PCR Mutagenesis Reveals Functional Domains of a Bacterial Transcriptional Activator, TraJ
Article Snippet: The following Escherichia coli strains were used: ED24 (F− Lac− Spcr ) , MC4100 [F− araD139 Δ( argF-lac ) U169 rpsL150 (Smr ) relA1 flb5301 deoC1 ptsF25 rbsR ] , DH5α [F− Δ lacU169 ϕ80d lacZ ΔM15 supE44 hsdR17 recA1 endA1 gyrA96 (Nalr ) thi-1 relA1 ] , BL21(DE3) [F− ompT hsdS B (rB − mB − ) gal dcm ] (Invitrogen), Mach1-T1R [F− ϕ80d lacZ ΔM15 Δ lacX74 hsd R (rK − mK + ) Δ recA1398 endA1 tonA ] (Invitrogen), C600 (F− supE44 thi-1 thr-1 leuB lacy tonA21 ), and SG12064 (C600 hslV ::Cm) ( ).

Activity Assay:

Article Title: Characterization of Hemolytic Escherichia coli Strains in Ferrets: Recognition of Candidate Virulence Factor CNF1
Article Snippet: .. Bacterial sonicates or supernatants of seven ferret E. coli isolates and one nonpathogenic laboratory strain of E. coli without detectable cnf DNA homology or CNF activity, DH5α (Invitrogen, Carlsbad, Calif.), were evaluated for cytopathic effects on HeLa cell (CCL-2) monolayers. ..

Expressing:

Article Title: Genetic identification of potential RNA-binding regions in a group II intron-encoded reverse transcriptase
Article Snippet: .. BL21(DE3) and BL21(DE3)-R3/Rosetta (Novagen) were used for LtrA protein expression , and DH5α, DH10B, and OmniMAX 2-T1R cells (Invitrogen) were used for plasmid DNA preparation and cloning. ..

Article Title: Identification and Characterization of a New Enoyl Coenzyme A Hydratase Involved in Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates in Recombinant Escherichia coli
Article Snippet: .. E. coli XL1-Blue (Stratagene Cloning Systems, La Jolla, Calif.) and DH5α (Invitrogen Life Technologies, Carlsbad, Calif.) were used as host strains for general cloning works and gene expression. ..

Plasmid Preparation:

Article Title: Genetic identification of potential RNA-binding regions in a group II intron-encoded reverse transcriptase
Article Snippet: .. BL21(DE3) and BL21(DE3)-R3/Rosetta (Novagen) were used for LtrA protein expression , and DH5α, DH10B, and OmniMAX 2-T1R cells (Invitrogen) were used for plasmid DNA preparation and cloning. ..

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  • 96
    Thermo Fisher max efficiency dh5α competent cells
    Diagnostic PCR confirms the deletion of TK0566 from the T. <t>kodakarensis</t> genome The same primer pairs are used on the <t>TS559</t> genome as the deletion genome. The size shift using primers A/B corresponds to the deletion of TK0566 while the absence of products for the reactions using C/D and E/F confirms the gene has been deleted from its native locus. The absence of products using primers C/D demonstrates that TK0566 is not present anywhere in the genome of the deletion strain.
    Max Efficiency Dh5α Competent Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher e coli dh5α
    PCR amplification of E.coli <t>DH5α</t> clones Lane 2 to 7 –positive amplicons Lane1- DNA marker
    E Coli Dh5α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 139 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher luciferase expressing e coli dh5α paklux2
    ICE Mh1 PM22 fitness costs, addiction, and consequences for antimicrobial resistance in transconjugants. (A) Growth curves of P. multocida CCUG 17976 (spontaneous rifampin-resistant mutant) and isogenic ICE Mh1 PM22 transconjugant. Mean of 4 biological replicates with SEM. (B) Growth curves of M. haemolytica ATCC 33396 (spontaneous rifampin-resistant mutant) and isogenic ICE Mh1 PM22 transconjugant. Mean of 4 biological replicates with SEM. (C) Luciferase-based competition index from co-cultures of ICE Mh1 PM22 transconjugants and E. coli <t>DH5α</t> harboring the luciferase expression plasmid <t>pAKlux2.</t> Mean of 4 biological replicates with SEM; t-test, ∗ P ≤ 0.05. (D) Long-term repeated passage of ICE Mh1 PM22 transconjugants. Transconjugants were grown in MH (no drug) or MH + 0.5 MIC (subinhibitory; MIC for susceptible P. multocida CCUG 17976 or M. haemolytica ATCC 33396 WT) for 150 days and plated on media with or without selective concentrations of indicated antimicrobials (i.e., 0.5 MIC for non-susceptible isogenic transconjugants). Mean of 3 biological replicates with SEM. (E) Upper panel: growth curves of co-cultures of E. coli DH5α pAKlux2 and E. coli DH5α, P. multocida CCUG 17976 ICE Mh1 PM22 , or M. haemolytica ATCC 33396 ICE Mh1 PM22 in subinhibitory (0.5 MIC for susceptible P. multocida CCUG 17976 or M. haemolytica ATCC 33396) concentrations of oxytetracycline (left) or spectinomycin (right). Mean of 3 biological replicates with SEM. Lower panel: detection of E. coli luciferase production in co-cultures (as above) with either oxytetracycline (left) or spectinomycin (right). (F) Schematic of checkerboard synergy assay for drug interaction screening.
    Luciferase Expressing E Coli Dh5α Paklux2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Diagnostic PCR confirms the deletion of TK0566 from the T. kodakarensis genome The same primer pairs are used on the TS559 genome as the deletion genome. The size shift using primers A/B corresponds to the deletion of TK0566 while the absence of products for the reactions using C/D and E/F confirms the gene has been deleted from its native locus. The absence of products using primers C/D demonstrates that TK0566 is not present anywhere in the genome of the deletion strain.

    Journal: Bio-protocol

    Article Title: Markerless Gene Editing in the Hyperthermophilic Archaeon Thermococcus kodakarensis

    doi: 10.21769/BioProtoc.2604

    Figure Lengend Snippet: Diagnostic PCR confirms the deletion of TK0566 from the T. kodakarensis genome The same primer pairs are used on the TS559 genome as the deletion genome. The size shift using primers A/B corresponds to the deletion of TK0566 while the absence of products for the reactions using C/D and E/F confirms the gene has been deleted from its native locus. The absence of products using primers C/D demonstrates that TK0566 is not present anywhere in the genome of the deletion strain.

    Article Snippet: 1 ml TB syringe (BD, catalog number: 309624) 1.7 ml microcentrifuge tubes (VWR, catalog number: 490004-444) 0.2 ml PCR tubes (VWR, catalog number: 20170-012) Polystyrene Petri plates (Fisher Scientific, catalog number: S33580A) Split rubber stopper (DWK Life Sciences, Wheaton, catalog number: W224100-282) 20 mm aluminum seals (DWK Life Sciences, Wheaton, catalog number: 224178-01) 20 mm E-Z Crimper, Standard Seal (DWK Life Sciences, Wheaton, catalog number: W225303) 20 mm E-Z Decapper (DWK Life Sciences, Wheaton, catalog number: W225353) Polycarbonate centrifuge tubes (Beckman Coulter, catalog number: 361690) Cell spreader (Fisher Scientific, catalog number: 08-100-10) 10 ml serum bottles (DWK Life Sciences, Wheaton, catalog number: 223739) Face shields, lab coats, and autoclave gloves Paper towels T. kodakarensis strain TS559 ( ) DH5α E. coli competent cells (Thermo Fisher Scientific, Invitrogen™, catalog number: 18258012) XL1-Blue E. coli competent cells (Agilent Technologies, catalog number: 200228) pTS700 ( ) Note: Please contact corresponding author to obtain plasmid.

    Techniques: Diagnostic Assay, Polymerase Chain Reaction

    Overview of the markerless deletion scheme used in T. kodakarensis At the top of the figure is the B-plasmid used to delete the target gene from the genome. The plasmid recombines into the genome providing agmatine prototrophy to recipient cells and yields an intermediate genome. Two intermediate genomes are possible; however only one is depicted here. A second spontaneous recombination event excises plasmid sequences and permits survival in the presence of cytotoxic 6-MP. This second recombination event will result in the desired deletion genome (left) or the restoration of the TS559 genome (right).

    Journal: Bio-protocol

    Article Title: Markerless Gene Editing in the Hyperthermophilic Archaeon Thermococcus kodakarensis

    doi: 10.21769/BioProtoc.2604

    Figure Lengend Snippet: Overview of the markerless deletion scheme used in T. kodakarensis At the top of the figure is the B-plasmid used to delete the target gene from the genome. The plasmid recombines into the genome providing agmatine prototrophy to recipient cells and yields an intermediate genome. Two intermediate genomes are possible; however only one is depicted here. A second spontaneous recombination event excises plasmid sequences and permits survival in the presence of cytotoxic 6-MP. This second recombination event will result in the desired deletion genome (left) or the restoration of the TS559 genome (right).

    Article Snippet: 1 ml TB syringe (BD, catalog number: 309624) 1.7 ml microcentrifuge tubes (VWR, catalog number: 490004-444) 0.2 ml PCR tubes (VWR, catalog number: 20170-012) Polystyrene Petri plates (Fisher Scientific, catalog number: S33580A) Split rubber stopper (DWK Life Sciences, Wheaton, catalog number: W224100-282) 20 mm aluminum seals (DWK Life Sciences, Wheaton, catalog number: 224178-01) 20 mm E-Z Crimper, Standard Seal (DWK Life Sciences, Wheaton, catalog number: W225303) 20 mm E-Z Decapper (DWK Life Sciences, Wheaton, catalog number: W225353) Polycarbonate centrifuge tubes (Beckman Coulter, catalog number: 361690) Cell spreader (Fisher Scientific, catalog number: 08-100-10) 10 ml serum bottles (DWK Life Sciences, Wheaton, catalog number: 223739) Face shields, lab coats, and autoclave gloves Paper towels T. kodakarensis strain TS559 ( ) DH5α E. coli competent cells (Thermo Fisher Scientific, Invitrogen™, catalog number: 18258012) XL1-Blue E. coli competent cells (Agilent Technologies, catalog number: 200228) pTS700 ( ) Note: Please contact corresponding author to obtain plasmid.

    Techniques: Plasmid Preparation

    PCR amplification of E.coli DH5α clones Lane 2 to 7 –positive amplicons Lane1- DNA marker

    Journal: Veterinary World

    Article Title: Cloning and sequence analysis of a partial CDS of leptospiral ligA gene in pET-32a – Escherichia coli DH5α system

    doi: 10.14202/vetworld.2018.557-561

    Figure Lengend Snippet: PCR amplification of E.coli DH5α clones Lane 2 to 7 –positive amplicons Lane1- DNA marker

    Article Snippet: The pET-32a ligA plasmid construct was extracted from the transformed E. coli DH5α using Thermo Scientific Gene JET plasmid Miniprep Kit and subjected to sequencing.

    Techniques: Polymerase Chain Reaction, Amplification, Clone Assay, Marker

    ICE Mh1 PM22 fitness costs, addiction, and consequences for antimicrobial resistance in transconjugants. (A) Growth curves of P. multocida CCUG 17976 (spontaneous rifampin-resistant mutant) and isogenic ICE Mh1 PM22 transconjugant. Mean of 4 biological replicates with SEM. (B) Growth curves of M. haemolytica ATCC 33396 (spontaneous rifampin-resistant mutant) and isogenic ICE Mh1 PM22 transconjugant. Mean of 4 biological replicates with SEM. (C) Luciferase-based competition index from co-cultures of ICE Mh1 PM22 transconjugants and E. coli DH5α harboring the luciferase expression plasmid pAKlux2. Mean of 4 biological replicates with SEM; t-test, ∗ P ≤ 0.05. (D) Long-term repeated passage of ICE Mh1 PM22 transconjugants. Transconjugants were grown in MH (no drug) or MH + 0.5 MIC (subinhibitory; MIC for susceptible P. multocida CCUG 17976 or M. haemolytica ATCC 33396 WT) for 150 days and plated on media with or without selective concentrations of indicated antimicrobials (i.e., 0.5 MIC for non-susceptible isogenic transconjugants). Mean of 3 biological replicates with SEM. (E) Upper panel: growth curves of co-cultures of E. coli DH5α pAKlux2 and E. coli DH5α, P. multocida CCUG 17976 ICE Mh1 PM22 , or M. haemolytica ATCC 33396 ICE Mh1 PM22 in subinhibitory (0.5 MIC for susceptible P. multocida CCUG 17976 or M. haemolytica ATCC 33396) concentrations of oxytetracycline (left) or spectinomycin (right). Mean of 3 biological replicates with SEM. Lower panel: detection of E. coli luciferase production in co-cultures (as above) with either oxytetracycline (left) or spectinomycin (right). (F) Schematic of checkerboard synergy assay for drug interaction screening.

    Journal: Frontiers in Microbiology

    Article Title: Emerging Variants of the Integrative and Conjugant Element ICEMh1 in Livestock Pathogens: Structural Insights, Potential Host Range, and Implications for Bacterial Fitness and Antimicrobial Therapy

    doi: 10.3389/fmicb.2019.02608

    Figure Lengend Snippet: ICE Mh1 PM22 fitness costs, addiction, and consequences for antimicrobial resistance in transconjugants. (A) Growth curves of P. multocida CCUG 17976 (spontaneous rifampin-resistant mutant) and isogenic ICE Mh1 PM22 transconjugant. Mean of 4 biological replicates with SEM. (B) Growth curves of M. haemolytica ATCC 33396 (spontaneous rifampin-resistant mutant) and isogenic ICE Mh1 PM22 transconjugant. Mean of 4 biological replicates with SEM. (C) Luciferase-based competition index from co-cultures of ICE Mh1 PM22 transconjugants and E. coli DH5α harboring the luciferase expression plasmid pAKlux2. Mean of 4 biological replicates with SEM; t-test, ∗ P ≤ 0.05. (D) Long-term repeated passage of ICE Mh1 PM22 transconjugants. Transconjugants were grown in MH (no drug) or MH + 0.5 MIC (subinhibitory; MIC for susceptible P. multocida CCUG 17976 or M. haemolytica ATCC 33396 WT) for 150 days and plated on media with or without selective concentrations of indicated antimicrobials (i.e., 0.5 MIC for non-susceptible isogenic transconjugants). Mean of 3 biological replicates with SEM. (E) Upper panel: growth curves of co-cultures of E. coli DH5α pAKlux2 and E. coli DH5α, P. multocida CCUG 17976 ICE Mh1 PM22 , or M. haemolytica ATCC 33396 ICE Mh1 PM22 in subinhibitory (0.5 MIC for susceptible P. multocida CCUG 17976 or M. haemolytica ATCC 33396) concentrations of oxytetracycline (left) or spectinomycin (right). Mean of 3 biological replicates with SEM. Lower panel: detection of E. coli luciferase production in co-cultures (as above) with either oxytetracycline (left) or spectinomycin (right). (F) Schematic of checkerboard synergy assay for drug interaction screening.

    Article Snippet: For 2-strain co-culture experiments, luciferase-expressing E. coli DH5α pAKlux2 and test strains (including DH5α without pAKlux2 as a control) were each inoculated at an OD600 of 0.0025 (i.e., total OD600 of ∼0.005) into black clear bottom 96-well plates (Nunc, Thermo-Fisher Scientific, Ottawa, ON, Canada).

    Techniques: Mutagenesis, Luciferase, Expressing, Plasmid Preparation