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  • 99
    ATCC e coli dh5α
    ( a ) The PCA results of E. coli <t>DH5α</t> incubated under different GO concentrations. 1 represents the control group and 2–6 principal components are from the samples incubated with different GO concentrations at 5, 20, 40, 80 and 120 μg/mL for 2 h. ( d ) The PCA results of time experiment. 1 represents the control group. 2–6 represent principal components obtained from the data incubated with GO (80 μg/mL) at different time: 0, 1, 2, 3 and 4 h. ( b , e ) show the relationship between GO concentration or incubation time and the parameter D as well as the cell viability rate of E. coli DH5α. ( c , f ) present the bivariate analysis results, and a significant negative correlation between the parameter D and cell viability rate were obtained in concentration experiment (Person’s correlation coefficient: −0.99, p
    E Coli Dh5α, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 331 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dh5α competent cells
    ATRX overexpression plasmids contain IS10 insertions. (A) Schematic map of the insertions found in IS10-GFP-ATRX and IS10-ATRX-YFP. The exon structure (alternate white and gray bars) of ATRX cDNA isoform 2 (top) and some relevant domains from the protein sequence (bottom) are shown. The insertion sites are denoted by black bars and their positions in the cDNA and the protein are described in the right box. Black arrows show the direction of the IS10 elements found. The stop codons introduced by IS10 in the protein sequence are indicated with asterisks. The position of two of the primer pairs used for analyzing ATRX sequence is shown (white and gray arrows). The NLS within ATRX cDNA is indicated with a black bar. (B) PCR analysis of IS10-GFP-ATRX, IS10-ATRX-YFP and IF-GFP-ATRX using the primers shown in (A). Both amplicons of IF-GFP-ATRX have the expected size (1,449 bp for amplicon I and 1,935 for amplicon II) while amplicon I shows an insertion in IS10-ATRX-YFP and amplicon II shows an insertion in IS10-GFP-ATRX (both have additional 1,336 bp). (C) Representative EcoRI digestion patterns of the IF-GFP-ATRX plasmid grown in two different bacteria strains. The Stbl4-derived plasmid has the expected pattern while the plasmid derived from <t>DH5α</t> shows an insertion in the 2,400bp fragment (shift from a 2,468 bp fragment to ~3,800 bp).
    Dh5α Competent Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2145 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher e coli dh5α
    Confirmation of the intracellular location of ChiA74∆s p in Escherichia coli and Bacillus thuringiensis 4Q7 using a chimeric construct with the green fluorescent protein (GFP) gene. (A) Schematic illustration that shows the fusion of chiA74 ∆s p with gfp . Panel 1, two constructs were developed, under regulation of the chiA74 wildtype promoter (wp) or under control of the cytA - p/ STAB-SD system. Lollipop indicates transcriptional terminator. Oligonucleotides used to make chimeric construct with gfp are shown in Table 1 . Panel 2, confirmation of chimeric constructs by PCR, primers used for amplification are showed in parenthesis and in Table 1 . L, 1 kb (kilobase) DNA Ladder (New England Biolabs); Lane 1, pEHchiA74∆ sp - gfp (primers: chiA74-1, chiA74-3); lane 2, pEHchiA74∆ sp - gfp (primers: chiA74-1, gfp-3); lane 3, pEHchiA74∆ sp - gfp (primers: gfp-1, chiA74-4); lane 4, pEB chi A74∆sp- gfp (primers: cytSTAB-1, chiA74-4); Lane 5, pEB chi A74∆sp- gfp (primers: cytSTAB-1, gfp-3); lane 6, pEB chi A74∆sp- gfp (primers: gfp-1, chiA74-4). (B) Phase contrast (left) and fluorescence micrographs (right) of recombinant strains of E. coli and B. thuringiensis strain 4Q7 expressing the chimeric protein ChiA74∆sp-GFP. Panel 1, E. coli- pEHchiA74∆ sp - gfp ; panel 2, 4Q7 - pEHchiA74∆ sp - gfp ; panel 3, E. coli- pEBchiA74∆ sp - gfp ; panel 4, 4Q7 - pEBchiA74∆ sp - gfp ; panel 5, E. coli <t>DH5α;</t> panel 6, 4Q7. Samples of recombinant strains of B. thuringiensis were collected at ~ 72 h.
    E Coli Dh5α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 3652 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher e coli strain dh5α
    ΦTE-F expresses ToxI RNA during infection. (A) Upper panel; an S1-nuclease protection assay was used to detect ToxI levels from a ToxIN plasmid during ΦTE wt infection, using an antisense probe against the full 5.5 repeat ToxI sequence [46] . The antisense ToxI-probe was first hybridised to 10 µg of total RNA prepared from Pba ToxIN (pMJ4) at different times after ΦTE infection, and then followed by S1-nuclease treatment. Numbers (+) indicate the time (min) after infection or (−) without the addition of phage. Pba ToxIN-FS (pTA47) and Pba serve as positive and negative controls, respectively. A non-hybridized S1-digested probe (+S1) serves as a further negative control. <t>DH5α</t> 1.5 repeats (pTA96), a non-S1 digested probe (−S1) and an in vitro transcribed Hammerhead ribozyme (HHRz), which cleaves itself during transcription, serve as size markers. HHRz was prepared as described previously [45] . Lower panel; Western blot targeting C-terminal FLAG tagged ToxN contained within total protein harvested from Pba ToxIN (pMJ4) at different time points, with (+, left) and without (−, right) phage infection. Time 0 indicates a sample taken immediately after infection. Total protein from Pba ToxIN (pMJ4) (−) serves as positive control. (B) Infection with escape phage ΦTE-F. Levels of ToxI were determined by S1-assay (upper) as described in (A) with and without infection. ToxN levels were estimated by Western blotting (lower) as described in (A). (C) Expression of the ΦTE-F ToxI locus. An S1-nuclease assay targeting ToxI was performed on total RNA of Pba (pBR322) at different times during ΦTE-F infection. Pba ToxIN (pMJ4) and DH5α serve as positive and negative controls, respectively.
    E Coli Strain Dh5α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 939 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dh5α
    E. coli <t>DH5α</t> cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.
    Dh5α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 2454 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore e coli dh5α
    Comparison of bioluminescence from S. aureus and E. coli containing the native luxCDABE operon versus that in strains containing the modified luxABCDE operon. Exponential cultures of S. aureus RN4220 pMK4 luxABCDE P1 (-■-), S. aureus RN4220 pMK4 luxCDABE P1 (-▴-), E. coli <t>DH5α</t> pMK4 luxABCDE P1 (‥■‥) and E. coli DH5α pMK4 luxCDABE P1 (‥▴‥) were diluted across black 96-well microtiter plates in doubling dilutions (−0.3 log) and monitored for light over a period of 30 min using the ICCD camera. The content of each well was then plated to allow the number of CFU to be compared to the level of bioluminescence (RLU).
    E Coli Dh5α, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 769 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher e coli dh5α competent cells
    Immunoprecipitation to determine the specificity of DivIVA interactions. (A) Immunoprecipitation of GFP-DivIVA-cI-Spo0J complex (lane 1), GFP-Spo0J-cI-DivIVA complex (lane 2), GFP-FtsZ-cI-DivIVA complex (lane 3), and GFP-DivIVA-cI-FtsZ complex (lane 4). (B) Immunoprecipitation of cI-DivIVA-GFP-PrfA complex (lane 1). Protein extracts were immunoprecipitated with anti-GFP antibodies and were detected with anti-cI rabbit polyclonal antibodies by Western blotting as described in Materials and Methods. Lane M contained molecular weight markers. In panel B lane 2 contained crude extract of E. coli strain <t>DH5α</t> expressing cI-DivIVA, detected with anti-cI antibodies.
    E Coli Dh5α Competent Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 498 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher multishot stripwell dh5α t1r competent cells
    YegI is an integral membrane protein A) Predicted topology of YegI using Phobius. Amino acid positions at which phoA-lacZα reporters are fused are indicated along with associated phenotypes. Phenotypes of phoAlacZα fusions due to LacZ activity are denoted as red ellipses while PhoA activity are denoted as blue ellipses. Predicted transmembrane domains (I and II) are depicted as grey rectangles. (B) Membrane topology analysis: <t>DH5α</t> transformed with different phoAlacZα reporters were streaked on an LB plate containing 5-bromo-4-chloro-3-indolyl phosphate disodium salt (X-Phos, 80μg/ml), 6-chloro-3-indolyl-β-D-galactoside (Red-Gal, 100 μg/ml, 0.1 % w/v arabinose and 100 μg/ml of ampicillin. Control pBAD24: control strain expressing pBAD24 empty vector, control phoAlacZα: strain expressing phoAlacZα in pBAD24 without YegI. Amino acid positions at which phoAlacZα reporters are fused are indicated.
    Multishot Stripwell Dh5α T1r Competent Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Stratagene e coli dh5α
    Strategy for gene replacement in Streptomyces . ( A )IV (Apra R ), oriT of RK2, and two FRT sites was amplified by PCR by using primers containing 39-nt 5′ homology extensions corresponding to regions flanking the S. coelicolor target gene (dotted lines a and b) and 20-nt 3′ homology to the unique priming sites P1 and P2 of the disruption cassette. ( B ) The PCR product from A was used to transform E. coli BW25113/pIJ790 (expressing the λ-Red recombination functions), containing the Supercos-1 cosmid with the cloned target gene ( cyc2 ). ( C ) Apra R transformants were selected, and the recombinant cosmid was identified by PCR and restriction analysis. ( D ) The potent methyl-specific restriction system of S. coelicolor was circumvented by introducing the recombinant cosmid into the methylation-deficient E. coli host ET12567/pUZ8002. The cosmid was mobilized in trans by pUZ8002 to Streptomyces by conjugation. ( E ) Selected exconjugants (Apra R ) were screened for Kan S (loss of the Kan resistance gene neo ), and the double cross-over allelic exchange was confirmed by PCR and Southern blot analysis. ( F ) E. coli <t>DH5α</t> cells containing pCP20 were transformed with the mutagenized cosmid DNA, and FLP synthesis was induced. ( G) The loss of the disruption resistance marker was tested, and the cosmid was introduced by polyethylene glycol (PEG)-mediated transformation into the S. coelicolor mutant carrying a marked deletion within the gene of interest. ( H ) Kan R transformants arising from single cross-over events were restreaked nonselectively and screened for the loss of both Kan R and Apra R , indicating a successful replacement by the in-frame deletion marked only by a “scar” sequence of 81 bp containing priming sites P1 and P2 (underlined), and one FRT site.
    E Coli Dh5α, supplied by Stratagene, used in various techniques. Bioz Stars score: 93/100, based on 446 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher subcloning efficiency dh5α competent cells
    D1 anaerobic protein purity. Protein was expressed in <t>DH5α</t> from the pASG-IBA35 vector A. Steps of D1 anaerobic purification process. Numbers above the gel mark fractions: 1 – before induction, 2- after induction, 3 – lysate, 4- pellet after centrifugation at 40 000 rcf, 5- NiNTA flow through, 6 – elution (this D1 anaerobic protein batch was used for EXAFS experiments). The marked band with mass 30 kDa corresponds to D1 protein. B. The purity of D1 anaerobic protein batch used for TSA, crystallization and SEC HPLC analysis (lanes 1 and 2).
    Subcloning Efficiency Dh5α Competent Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 583 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad e coli dh5α
    H. pylori produces a luxS -dependent AI-2 activity. Cell-free supernatants were tested for the ability to induce luminescence expression in V. harveyi BB170. Ten percent cell-free supernatants or sterile media were mixed with the reporter strain in microtiter plates and incubated at 30°C on a rotary shaker. Aliquots were taken, and both cell density and light production were determined. Activity is reported as fold activation of luminescence of BB170 over the level of luminescence when sterile media were added. Assays were repeated at least two times. (A) Wild-type H. pylori and Typhimurium 14028 supernatants contain signaling substances that induce expression of luminescence in V. harveyi BB170, while the luxS deletion strain EJ103 does not. (B) H. pylori luxS expression in E. coli <t>DH5α</t> restores AI-2 activity. The error bars indicate standard deviations.
    E Coli Dh5α, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 410 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co e coli dh5α
    Dynamic monitoring of intestinal epithelial cell responses to intestinal bacteria. HT-29 cells (10,000 cells per well) were seeded into E-plates. The next day, cells were infected with Y. enterocolitica (A), S. flexneri (B), L. monocytogenes (C), E. coli <t>DH5α</t> (E), and Lactobacillus (F). Arrows, bacterial addition. Representative curves are an average of four replicate wells.
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    Difco e coli dh5α
    Efficiency of electroporation of pTOPO/FNL10 and pFNLTP1 into E. coli <t>DH5α</t> or Francisella spp. ( F. tularensis LVS or F. novicida U112). The transformation efficiency, expressed as log CFU per microgram of DNA (mean ± standard deviation), was determined from three individual preparations of competent cells. Competent cells of E. coli were prepared with 10% glycerol, while 0.5 M sucrose was used to prepare competent cells of F. tularensis LVS and F. novicida U112. Most electroporations were performed with 100 ng of plasmid DNA; the only exception was the electroporation of pTOPO/FNL10 into F. tularensis LVS, for which 500 ng was used. The strains indicated in parentheses are the hosts from which plasmid DNA was isolated.
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    TransGen biotech co escherichia coli dh5α
    Efficiency of electroporation of pTOPO/FNL10 and pFNLTP1 into E. coli <t>DH5α</t> or Francisella spp. ( F. tularensis LVS or F. novicida U112). The transformation efficiency, expressed as log CFU per microgram of DNA (mean ± standard deviation), was determined from three individual preparations of competent cells. Competent cells of E. coli were prepared with 10% glycerol, while 0.5 M sucrose was used to prepare competent cells of F. tularensis LVS and F. novicida U112. Most electroporations were performed with 100 ng of plasmid DNA; the only exception was the electroporation of pTOPO/FNL10 into F. tularensis LVS, for which 500 ng was used. The strains indicated in parentheses are the hosts from which plasmid DNA was isolated.
    Escherichia Coli Dh5α, supplied by TransGen biotech co, used in various techniques. Bioz Stars score: 93/100, based on 155 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene escherichia coli strains dh5α
    Efficiency of electroporation of pTOPO/FNL10 and pFNLTP1 into E. coli <t>DH5α</t> or Francisella spp. ( F. tularensis LVS or F. novicida U112). The transformation efficiency, expressed as log CFU per microgram of DNA (mean ± standard deviation), was determined from three individual preparations of competent cells. Competent cells of E. coli were prepared with 10% glycerol, while 0.5 M sucrose was used to prepare competent cells of F. tularensis LVS and F. novicida U112. Most electroporations were performed with 100 ng of plasmid DNA; the only exception was the electroporation of pTOPO/FNL10 into F. tularensis LVS, for which 500 ng was used. The strains indicated in parentheses are the hosts from which plasmid DNA was isolated.
    Escherichia Coli Strains Dh5α, supplied by Stratagene, used in various techniques. Bioz Stars score: 91/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher library efficiency dh5α competent cells
    Efficiency of electroporation of pTOPO/FNL10 and pFNLTP1 into E. coli <t>DH5α</t> or Francisella spp. ( F. tularensis LVS or F. novicida U112). The transformation efficiency, expressed as log CFU per microgram of DNA (mean ± standard deviation), was determined from three individual preparations of competent cells. Competent cells of E. coli were prepared with 10% glycerol, while 0.5 M sucrose was used to prepare competent cells of F. tularensis LVS and F. novicida U112. Most electroporations were performed with 100 ng of plasmid DNA; the only exception was the electroporation of pTOPO/FNL10 into F. tularensis LVS, for which 500 ng was used. The strains indicated in parentheses are the hosts from which plasmid DNA was isolated.
    Library Efficiency Dh5α Competent Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 356 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ( a ) The PCA results of E. coli DH5α incubated under different GO concentrations. 1 represents the control group and 2–6 principal components are from the samples incubated with different GO concentrations at 5, 20, 40, 80 and 120 μg/mL for 2 h. ( d ) The PCA results of time experiment. 1 represents the control group. 2–6 represent principal components obtained from the data incubated with GO (80 μg/mL) at different time: 0, 1, 2, 3 and 4 h. ( b , e ) show the relationship between GO concentration or incubation time and the parameter D as well as the cell viability rate of E. coli DH5α. ( c , f ) present the bivariate analysis results, and a significant negative correlation between the parameter D and cell viability rate were obtained in concentration experiment (Person’s correlation coefficient: −0.99, p

    Journal: Scientific Reports

    Article Title: Rapidly Probing Antibacterial Activity of Graphene Oxide by Mass Spectrometry-based Metabolite Fingerprinting

    doi: 10.1038/srep28045

    Figure Lengend Snippet: ( a ) The PCA results of E. coli DH5α incubated under different GO concentrations. 1 represents the control group and 2–6 principal components are from the samples incubated with different GO concentrations at 5, 20, 40, 80 and 120 μg/mL for 2 h. ( d ) The PCA results of time experiment. 1 represents the control group. 2–6 represent principal components obtained from the data incubated with GO (80 μg/mL) at different time: 0, 1, 2, 3 and 4 h. ( b , e ) show the relationship between GO concentration or incubation time and the parameter D as well as the cell viability rate of E. coli DH5α. ( c , f ) present the bivariate analysis results, and a significant negative correlation between the parameter D and cell viability rate were obtained in concentration experiment (Person’s correlation coefficient: −0.99, p

    Article Snippet: Principal component analysis of metabolite fingerprinting of E. coli DH5α and ATCC 25922 after incubation with GO at different conditions shows metabolic disturbance of these bacteria ( a,d and a,b).

    Techniques: Incubation, Concentration Assay

    ( a ) The three principal components in PCA scores plot represent the E coli. DH5α control group (◾), GO group (▴) and AgNPs (▾) group (5 μg/mL, 2 h). ( b ) The three principal components in PCA scores plot represent the E coli. DH5α control group (◾), GO group (▴) and hydroxylated fullerene group (⦁) (80 μg/mL 2 h). ( c ) The three principal components in PCA scores plot represent the control group (▾), and the GO groups (▴) and hexagonal boron nitride (♦) group (80 μg/mL, 2 h). ( d–f ) The three principal components in PCA scores plot of ATCC 25922 represent the control group (◾), the GO group (▴) and AgNPs (▾) group (5 μg/mL, 2 h), hydroxylated fullerene group (⦁) (80 μg/mL 2 h) and hexagonal boron nitride (♦) incubation (80 μg/mL, 2 h), respectively.

    Journal: Scientific Reports

    Article Title: Rapidly Probing Antibacterial Activity of Graphene Oxide by Mass Spectrometry-based Metabolite Fingerprinting

    doi: 10.1038/srep28045

    Figure Lengend Snippet: ( a ) The three principal components in PCA scores plot represent the E coli. DH5α control group (◾), GO group (▴) and AgNPs (▾) group (5 μg/mL, 2 h). ( b ) The three principal components in PCA scores plot represent the E coli. DH5α control group (◾), GO group (▴) and hydroxylated fullerene group (⦁) (80 μg/mL 2 h). ( c ) The three principal components in PCA scores plot represent the control group (▾), and the GO groups (▴) and hexagonal boron nitride (♦) group (80 μg/mL, 2 h). ( d–f ) The three principal components in PCA scores plot of ATCC 25922 represent the control group (◾), the GO group (▴) and AgNPs (▾) group (5 μg/mL, 2 h), hydroxylated fullerene group (⦁) (80 μg/mL 2 h) and hexagonal boron nitride (♦) incubation (80 μg/mL, 2 h), respectively.

    Article Snippet: Principal component analysis of metabolite fingerprinting of E. coli DH5α and ATCC 25922 after incubation with GO at different conditions shows metabolic disturbance of these bacteria ( a,d and a,b).

    Techniques: Incubation

    ATRX overexpression plasmids contain IS10 insertions. (A) Schematic map of the insertions found in IS10-GFP-ATRX and IS10-ATRX-YFP. The exon structure (alternate white and gray bars) of ATRX cDNA isoform 2 (top) and some relevant domains from the protein sequence (bottom) are shown. The insertion sites are denoted by black bars and their positions in the cDNA and the protein are described in the right box. Black arrows show the direction of the IS10 elements found. The stop codons introduced by IS10 in the protein sequence are indicated with asterisks. The position of two of the primer pairs used for analyzing ATRX sequence is shown (white and gray arrows). The NLS within ATRX cDNA is indicated with a black bar. (B) PCR analysis of IS10-GFP-ATRX, IS10-ATRX-YFP and IF-GFP-ATRX using the primers shown in (A). Both amplicons of IF-GFP-ATRX have the expected size (1,449 bp for amplicon I and 1,935 for amplicon II) while amplicon I shows an insertion in IS10-ATRX-YFP and amplicon II shows an insertion in IS10-GFP-ATRX (both have additional 1,336 bp). (C) Representative EcoRI digestion patterns of the IF-GFP-ATRX plasmid grown in two different bacteria strains. The Stbl4-derived plasmid has the expected pattern while the plasmid derived from DH5α shows an insertion in the 2,400bp fragment (shift from a 2,468 bp fragment to ~3,800 bp).

    Journal: SpringerPlus

    Article Title: The ATRX cDNA is prone to bacterial IS10 element insertions that alter its structure

    doi: 10.1186/2193-1801-3-222

    Figure Lengend Snippet: ATRX overexpression plasmids contain IS10 insertions. (A) Schematic map of the insertions found in IS10-GFP-ATRX and IS10-ATRX-YFP. The exon structure (alternate white and gray bars) of ATRX cDNA isoform 2 (top) and some relevant domains from the protein sequence (bottom) are shown. The insertion sites are denoted by black bars and their positions in the cDNA and the protein are described in the right box. Black arrows show the direction of the IS10 elements found. The stop codons introduced by IS10 in the protein sequence are indicated with asterisks. The position of two of the primer pairs used for analyzing ATRX sequence is shown (white and gray arrows). The NLS within ATRX cDNA is indicated with a black bar. (B) PCR analysis of IS10-GFP-ATRX, IS10-ATRX-YFP and IF-GFP-ATRX using the primers shown in (A). Both amplicons of IF-GFP-ATRX have the expected size (1,449 bp for amplicon I and 1,935 for amplicon II) while amplicon I shows an insertion in IS10-ATRX-YFP and amplicon II shows an insertion in IS10-GFP-ATRX (both have additional 1,336 bp). (C) Representative EcoRI digestion patterns of the IF-GFP-ATRX plasmid grown in two different bacteria strains. The Stbl4-derived plasmid has the expected pattern while the plasmid derived from DH5α shows an insertion in the 2,400bp fragment (shift from a 2,468 bp fragment to ~3,800 bp).

    Article Snippet: ATRX transformation and growth in different bacterial strains ElectroMAX™ Stbl4™ competent cells (Life Technologies, 11635-018) and DH5α competent cells (Life Technologies 18258-012) were transformed according to manufacturer’s indications with 50 ng of IF-GFP-ATRX.

    Techniques: Over Expression, Sequencing, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Derivative Assay

    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

    Confirmation of the intracellular location of ChiA74∆s p in Escherichia coli and Bacillus thuringiensis 4Q7 using a chimeric construct with the green fluorescent protein (GFP) gene. (A) Schematic illustration that shows the fusion of chiA74 ∆s p with gfp . Panel 1, two constructs were developed, under regulation of the chiA74 wildtype promoter (wp) or under control of the cytA - p/ STAB-SD system. Lollipop indicates transcriptional terminator. Oligonucleotides used to make chimeric construct with gfp are shown in Table 1 . Panel 2, confirmation of chimeric constructs by PCR, primers used for amplification are showed in parenthesis and in Table 1 . L, 1 kb (kilobase) DNA Ladder (New England Biolabs); Lane 1, pEHchiA74∆ sp - gfp (primers: chiA74-1, chiA74-3); lane 2, pEHchiA74∆ sp - gfp (primers: chiA74-1, gfp-3); lane 3, pEHchiA74∆ sp - gfp (primers: gfp-1, chiA74-4); lane 4, pEB chi A74∆sp- gfp (primers: cytSTAB-1, chiA74-4); Lane 5, pEB chi A74∆sp- gfp (primers: cytSTAB-1, gfp-3); lane 6, pEB chi A74∆sp- gfp (primers: gfp-1, chiA74-4). (B) Phase contrast (left) and fluorescence micrographs (right) of recombinant strains of E. coli and B. thuringiensis strain 4Q7 expressing the chimeric protein ChiA74∆sp-GFP. Panel 1, E. coli- pEHchiA74∆ sp - gfp ; panel 2, 4Q7 - pEHchiA74∆ sp - gfp ; panel 3, E. coli- pEBchiA74∆ sp - gfp ; panel 4, 4Q7 - pEBchiA74∆ sp - gfp ; panel 5, E. coli DH5α; panel 6, 4Q7. Samples of recombinant strains of B. thuringiensis were collected at ~ 72 h.

    Journal: Microbial Cell Factories

    Article Title: Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74?sp chitinase inclusions, Cry crystals and spores for applied use

    doi: 10.1186/1475-2859-13-15

    Figure Lengend Snippet: Confirmation of the intracellular location of ChiA74∆s p in Escherichia coli and Bacillus thuringiensis 4Q7 using a chimeric construct with the green fluorescent protein (GFP) gene. (A) Schematic illustration that shows the fusion of chiA74 ∆s p with gfp . Panel 1, two constructs were developed, under regulation of the chiA74 wildtype promoter (wp) or under control of the cytA - p/ STAB-SD system. Lollipop indicates transcriptional terminator. Oligonucleotides used to make chimeric construct with gfp are shown in Table 1 . Panel 2, confirmation of chimeric constructs by PCR, primers used for amplification are showed in parenthesis and in Table 1 . L, 1 kb (kilobase) DNA Ladder (New England Biolabs); Lane 1, pEHchiA74∆ sp - gfp (primers: chiA74-1, chiA74-3); lane 2, pEHchiA74∆ sp - gfp (primers: chiA74-1, gfp-3); lane 3, pEHchiA74∆ sp - gfp (primers: gfp-1, chiA74-4); lane 4, pEB chi A74∆sp- gfp (primers: cytSTAB-1, chiA74-4); Lane 5, pEB chi A74∆sp- gfp (primers: cytSTAB-1, gfp-3); lane 6, pEB chi A74∆sp- gfp (primers: gfp-1, chiA74-4). (B) Phase contrast (left) and fluorescence micrographs (right) of recombinant strains of E. coli and B. thuringiensis strain 4Q7 expressing the chimeric protein ChiA74∆sp-GFP. Panel 1, E. coli- pEHchiA74∆ sp - gfp ; panel 2, 4Q7 - pEHchiA74∆ sp - gfp ; panel 3, E. coli- pEBchiA74∆ sp - gfp ; panel 4, 4Q7 - pEBchiA74∆ sp - gfp ; panel 5, E. coli DH5α; panel 6, 4Q7. Samples of recombinant strains of B. thuringiensis were collected at ~ 72 h.

    Article Snippet: All constructs (Figure A) were propagated in E. coli DH5α [supE44, DlacU169 (F80lacZDM15 ) hsdR17 recA1 end A1 gyrA96 thi-1 relA1 ] (Invitrogen, Carlsbad CA, USA) and then used for transforming the acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7, (hereafter 4Q7), and B. thuringiensis subsp. kurstaki HD1 (hereafter HD1) (Bacillus Genetic Stock Center, Columbus, OH).

    Techniques: Construct, Polymerase Chain Reaction, Amplification, Fluorescence, Recombinant, Expressing

    Expression of ChiA74Δsp in Escherichia coli DH5α and Bacillus thuringiensis 4Q7. (A) Schematic illustration of the strategy used to delete the secretion signal peptide-encoding sequence (shown as a rectangle inside the open reading frame) of chiA74 to generate chiA74∆ s p. Two constructs were developed, the first under regulation of wildtype promoter (wp) and the second under control of the strong cytA-p /STAB-SD promoter system. Lollipop indicates the putative transcriptional terminator site. Oligonucleotide sequences used to delete the signal peptide-coding sequence are shown in Table 1 . (B) Evaluation of endochitinase activity using solubilized intracellular proteins produced in E. coli. Panel 1: Zymogram using 4-MU-(GlcNAc) 3 for detection. Lane 1, E. coli- pEHchiA74∆s p ; lane 2, without sample; lane 3, E. coli- pEBchiA74∆s p; lane 4, E. coli DH5α . Black arrow indicates the position of ChiA74∆s p in recombinant E. coli strains . Panel 2: (a) E. coli- pEHchiA74∆s p , (b) E. coli- pEBchiA74∆s p. No endochitinase activity was observed with E. coli DH5α . (C) Phase contrast microscopy of recombinant strains of B. thuringiensis . Panel 1, 4Q7; panel 2, 4Q7 - pEHchiA74∆s p ; panel 3, 4Q7 - pEBchiA74∆s p . Black arrows indicate the positions of ChiA74∆s p inclusions. (D) Endochitinase activity determined using solubilized intracellular proteins of recombinant strains of B. thuringiensis. Panel 1: lane 1, 4Q7; lane 2, 4Q7 - pEHchiA74∆s p ; lane 3, 4Q7 - pEBchiA74∆s p. Black arrow indicates the position of ChiA74∆s p in recombinant 4Q7 strains . Panel 2: (a) 4Q7 - pEHchiA74∆s p , (b) 4Q7 - pEBchiA74∆s p . Activity of recombinant bacteria was normalized with the residual intracellular endochitinase activity of 4Q7.

    Journal: Microbial Cell Factories

    Article Title: Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74?sp chitinase inclusions, Cry crystals and spores for applied use

    doi: 10.1186/1475-2859-13-15

    Figure Lengend Snippet: Expression of ChiA74Δsp in Escherichia coli DH5α and Bacillus thuringiensis 4Q7. (A) Schematic illustration of the strategy used to delete the secretion signal peptide-encoding sequence (shown as a rectangle inside the open reading frame) of chiA74 to generate chiA74∆ s p. Two constructs were developed, the first under regulation of wildtype promoter (wp) and the second under control of the strong cytA-p /STAB-SD promoter system. Lollipop indicates the putative transcriptional terminator site. Oligonucleotide sequences used to delete the signal peptide-coding sequence are shown in Table 1 . (B) Evaluation of endochitinase activity using solubilized intracellular proteins produced in E. coli. Panel 1: Zymogram using 4-MU-(GlcNAc) 3 for detection. Lane 1, E. coli- pEHchiA74∆s p ; lane 2, without sample; lane 3, E. coli- pEBchiA74∆s p; lane 4, E. coli DH5α . Black arrow indicates the position of ChiA74∆s p in recombinant E. coli strains . Panel 2: (a) E. coli- pEHchiA74∆s p , (b) E. coli- pEBchiA74∆s p. No endochitinase activity was observed with E. coli DH5α . (C) Phase contrast microscopy of recombinant strains of B. thuringiensis . Panel 1, 4Q7; panel 2, 4Q7 - pEHchiA74∆s p ; panel 3, 4Q7 - pEBchiA74∆s p . Black arrows indicate the positions of ChiA74∆s p inclusions. (D) Endochitinase activity determined using solubilized intracellular proteins of recombinant strains of B. thuringiensis. Panel 1: lane 1, 4Q7; lane 2, 4Q7 - pEHchiA74∆s p ; lane 3, 4Q7 - pEBchiA74∆s p. Black arrow indicates the position of ChiA74∆s p in recombinant 4Q7 strains . Panel 2: (a) 4Q7 - pEHchiA74∆s p , (b) 4Q7 - pEBchiA74∆s p . Activity of recombinant bacteria was normalized with the residual intracellular endochitinase activity of 4Q7.

    Article Snippet: All constructs (Figure A) were propagated in E. coli DH5α [supE44, DlacU169 (F80lacZDM15 ) hsdR17 recA1 end A1 gyrA96 thi-1 relA1 ] (Invitrogen, Carlsbad CA, USA) and then used for transforming the acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7, (hereafter 4Q7), and B. thuringiensis subsp. kurstaki HD1 (hereafter HD1) (Bacillus Genetic Stock Center, Columbus, OH).

    Techniques: Expressing, Sequencing, Construct, Activity Assay, Produced, Recombinant, Microscopy

    ΦTE-F expresses ToxI RNA during infection. (A) Upper panel; an S1-nuclease protection assay was used to detect ToxI levels from a ToxIN plasmid during ΦTE wt infection, using an antisense probe against the full 5.5 repeat ToxI sequence [46] . The antisense ToxI-probe was first hybridised to 10 µg of total RNA prepared from Pba ToxIN (pMJ4) at different times after ΦTE infection, and then followed by S1-nuclease treatment. Numbers (+) indicate the time (min) after infection or (−) without the addition of phage. Pba ToxIN-FS (pTA47) and Pba serve as positive and negative controls, respectively. A non-hybridized S1-digested probe (+S1) serves as a further negative control. DH5α 1.5 repeats (pTA96), a non-S1 digested probe (−S1) and an in vitro transcribed Hammerhead ribozyme (HHRz), which cleaves itself during transcription, serve as size markers. HHRz was prepared as described previously [45] . Lower panel; Western blot targeting C-terminal FLAG tagged ToxN contained within total protein harvested from Pba ToxIN (pMJ4) at different time points, with (+, left) and without (−, right) phage infection. Time 0 indicates a sample taken immediately after infection. Total protein from Pba ToxIN (pMJ4) (−) serves as positive control. (B) Infection with escape phage ΦTE-F. Levels of ToxI were determined by S1-assay (upper) as described in (A) with and without infection. ToxN levels were estimated by Western blotting (lower) as described in (A). (C) Expression of the ΦTE-F ToxI locus. An S1-nuclease assay targeting ToxI was performed on total RNA of Pba (pBR322) at different times during ΦTE-F infection. Pba ToxIN (pMJ4) and DH5α serve as positive and negative controls, respectively.

    Journal: PLoS Genetics

    Article Title: Viral Evasion of a Bacterial Suicide System by RNA-Based Molecular Mimicry Enables Infectious Altruism

    doi: 10.1371/journal.pgen.1003023

    Figure Lengend Snippet: ΦTE-F expresses ToxI RNA during infection. (A) Upper panel; an S1-nuclease protection assay was used to detect ToxI levels from a ToxIN plasmid during ΦTE wt infection, using an antisense probe against the full 5.5 repeat ToxI sequence [46] . The antisense ToxI-probe was first hybridised to 10 µg of total RNA prepared from Pba ToxIN (pMJ4) at different times after ΦTE infection, and then followed by S1-nuclease treatment. Numbers (+) indicate the time (min) after infection or (−) without the addition of phage. Pba ToxIN-FS (pTA47) and Pba serve as positive and negative controls, respectively. A non-hybridized S1-digested probe (+S1) serves as a further negative control. DH5α 1.5 repeats (pTA96), a non-S1 digested probe (−S1) and an in vitro transcribed Hammerhead ribozyme (HHRz), which cleaves itself during transcription, serve as size markers. HHRz was prepared as described previously [45] . Lower panel; Western blot targeting C-terminal FLAG tagged ToxN contained within total protein harvested from Pba ToxIN (pMJ4) at different time points, with (+, left) and without (−, right) phage infection. Time 0 indicates a sample taken immediately after infection. Total protein from Pba ToxIN (pMJ4) (−) serves as positive control. (B) Infection with escape phage ΦTE-F. Levels of ToxI were determined by S1-assay (upper) as described in (A) with and without infection. ToxN levels were estimated by Western blotting (lower) as described in (A). (C) Expression of the ΦTE-F ToxI locus. An S1-nuclease assay targeting ToxI was performed on total RNA of Pba (pBR322) at different times during ΦTE-F infection. Pba ToxIN (pMJ4) and DH5α serve as positive and negative controls, respectively.

    Article Snippet: All experiments were performed with E. coli strain DH5α (Gibco/BRL) or Pectobacterium atrosepticum SCRI1043 and derivatives thereof.

    Techniques: Infection, Plasmid Preparation, Sequencing, Negative Control, In Vitro, Western Blot, Positive Control, Expressing, Nuclease Assay

    Analysis of pseudo-ToxI as a potential antitoxin. (A) Alignment of the pseudo-ToxI and ToxI RNA sequences. Pseudo-ToxI nucleotides are coloured to match (B) and (C), with the green and purple bases denoting the 5′ and 3′ ends of a single pseudoknot, respectively. Mutated nucleotides in pseudo-ToxI are coloured orange and numbered according to their grouping, whilst the asterisk indicates the additional 3′ nucleotide. The dotted line connecting the U in group 3 indicates the uracil that is deleted in the case of expanded repeats with 2T sequences rather than 3T. (B) Schematic of the ToxI pseudoknot. Each position containing a mutation in the pseudo-ToxI RNA has been bracketed, with the ToxI base separated from the pseudo-ToxI base by a ‘/’. The mutations have been grouped 1–5, according to position, and highlighted in orange, with the 5′ and 3′ termini in green and violet, respectively. Indels, such as U17 that is deleted in some pseudo-ToxI repeats, and the additional A* inserted in all, have been bordered by a dashed line. Base interactions are indicated by black lines, and duplex and triplex base-interactions are bordered in grey. (C) Detail of the ToxIN trimer with each pseudoknot shown either in blue, purple or beige. Each ToxN monomer is shown as a grey surface. The blue pseudoknot is oriented relative to (B). The positions of mutation groups are shown, with the group number encircled in the same colour as the corresponding pseudoknot. The additional nucleotide of group 5 is not visible as this was not in the original solved ToxIN structure. PDB: 2XDB. (D) Pseudo-ToxI cannot protect from ToxN in an over-expression assay. Protection assays were conducted as per Materials and Methods using strains of E. coli DH5α carrying both pTA49 (inducible ToxN) and a second inducible antitoxin vector as shown, including use of pTA100 as a vector-only control, “vector”. Error bars indicate the standard deviation of triplicate data. (E) Protection assays using mutants of ToxI carried out as in (D) with the antitoxin mutations in each construct numbered as per (B). (F) Protection assays carried out as in (D), testing the full escape loci of ΦTE wt, ΦTE-A and ΦTE-F with full ToxI as a positive control. Under these conditions, there was sufficient antitoxin present to inhibit induced ToxN even without specific induction of the ToxI and ΦTE-F constructs.

    Journal: PLoS Genetics

    Article Title: Viral Evasion of a Bacterial Suicide System by RNA-Based Molecular Mimicry Enables Infectious Altruism

    doi: 10.1371/journal.pgen.1003023

    Figure Lengend Snippet: Analysis of pseudo-ToxI as a potential antitoxin. (A) Alignment of the pseudo-ToxI and ToxI RNA sequences. Pseudo-ToxI nucleotides are coloured to match (B) and (C), with the green and purple bases denoting the 5′ and 3′ ends of a single pseudoknot, respectively. Mutated nucleotides in pseudo-ToxI are coloured orange and numbered according to their grouping, whilst the asterisk indicates the additional 3′ nucleotide. The dotted line connecting the U in group 3 indicates the uracil that is deleted in the case of expanded repeats with 2T sequences rather than 3T. (B) Schematic of the ToxI pseudoknot. Each position containing a mutation in the pseudo-ToxI RNA has been bracketed, with the ToxI base separated from the pseudo-ToxI base by a ‘/’. The mutations have been grouped 1–5, according to position, and highlighted in orange, with the 5′ and 3′ termini in green and violet, respectively. Indels, such as U17 that is deleted in some pseudo-ToxI repeats, and the additional A* inserted in all, have been bordered by a dashed line. Base interactions are indicated by black lines, and duplex and triplex base-interactions are bordered in grey. (C) Detail of the ToxIN trimer with each pseudoknot shown either in blue, purple or beige. Each ToxN monomer is shown as a grey surface. The blue pseudoknot is oriented relative to (B). The positions of mutation groups are shown, with the group number encircled in the same colour as the corresponding pseudoknot. The additional nucleotide of group 5 is not visible as this was not in the original solved ToxIN structure. PDB: 2XDB. (D) Pseudo-ToxI cannot protect from ToxN in an over-expression assay. Protection assays were conducted as per Materials and Methods using strains of E. coli DH5α carrying both pTA49 (inducible ToxN) and a second inducible antitoxin vector as shown, including use of pTA100 as a vector-only control, “vector”. Error bars indicate the standard deviation of triplicate data. (E) Protection assays using mutants of ToxI carried out as in (D) with the antitoxin mutations in each construct numbered as per (B). (F) Protection assays carried out as in (D), testing the full escape loci of ΦTE wt, ΦTE-A and ΦTE-F with full ToxI as a positive control. Under these conditions, there was sufficient antitoxin present to inhibit induced ToxN even without specific induction of the ToxI and ΦTE-F constructs.

    Article Snippet: All experiments were performed with E. coli strain DH5α (Gibco/BRL) or Pectobacterium atrosepticum SCRI1043 and derivatives thereof.

    Techniques: Mutagenesis, Over Expression, Plasmid Preparation, Standard Deviation, Construct, Positive Control

    E. coli DH5α cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.

    Journal: Nucleic Acids Research

    Article Title: Functional replacement of the endogenous tyrosyl-tRNA synthetase-tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion

    doi: 10.1093/nar/gkq080

    Figure Lengend Snippet: E. coli DH5α cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.

    Article Snippet: Strains, growth conditions, growth rate, reagents and non-natural amino acids Escherichia coli strains TOP10, BL21(DE3) and DH5α were purchased from Invitrogen, Novagen and Toyobo (Tokyo, Japan), respectively.

    Techniques: Expressing, Plasmid Preparation

    Evaluation of OmpT-mediated release and small-scale purification of displayed Affibody molecule. A . Flow-cytometric analysis for comparison of surface expression of recombinant proteins between p AraBAD -Z IgG -EC in OmpT-negative E. coli BL21 and p AraBAD -Z IgG -EC in OmpT-positive E. coli DH5α. B . SDS-PAGE showing IMAC-purified supernatants from p AraBAD -Z IgG -EC in OmpT-negative E. coli BL21 and p AraBAD -Z IgG -EC in OmpT-positive E. coli DH5α. Supernatants from non-induced samples, indicated with (−), were included as controls. C . Mass spectrum from ESI-MS analysis of IMAC-purified supernatants from p AraBAD -Z IgG -EC in OmpT-positive E. coli DH5α. Theoretical molecular weight of the OmpT-cleaved Z IgG -His 6 is 9712 Da. D . SPR-based biosensor analysis on the IMAC-purified Z IgG -His 6 . Response units (RU) on the y-axis and time on the x-axis. Representative sensorgrams from injection of Z IgG -His 6 at three different concentrations over human IgG immobilized on the chip surface. Injections were performed in duplicates.

    Journal: Microbial Cell Factories

    Article Title: An engineered autotransporter-based surface expression vector enables efficient display of Affibody molecules on OmpT-negative E. coli as well as protease-mediated secretion in OmpT-positive strains

    doi: 10.1186/s12934-014-0179-z

    Figure Lengend Snippet: Evaluation of OmpT-mediated release and small-scale purification of displayed Affibody molecule. A . Flow-cytometric analysis for comparison of surface expression of recombinant proteins between p AraBAD -Z IgG -EC in OmpT-negative E. coli BL21 and p AraBAD -Z IgG -EC in OmpT-positive E. coli DH5α. B . SDS-PAGE showing IMAC-purified supernatants from p AraBAD -Z IgG -EC in OmpT-negative E. coli BL21 and p AraBAD -Z IgG -EC in OmpT-positive E. coli DH5α. Supernatants from non-induced samples, indicated with (−), were included as controls. C . Mass spectrum from ESI-MS analysis of IMAC-purified supernatants from p AraBAD -Z IgG -EC in OmpT-positive E. coli DH5α. Theoretical molecular weight of the OmpT-cleaved Z IgG -His 6 is 9712 Da. D . SPR-based biosensor analysis on the IMAC-purified Z IgG -His 6 . Response units (RU) on the y-axis and time on the x-axis. Representative sensorgrams from injection of Z IgG -His 6 at three different concentrations over human IgG immobilized on the chip surface. Injections were performed in duplicates.

    Article Snippet: The plasmid and PCR fragment were ligated using a T4 DNA ligase (New England Biolabs), and transformed to E. coli DH5α cells (Invitrogen) by heat shock.

    Techniques: Purification, Flow Cytometry, Expressing, Recombinant, SDS Page, Mass Spectrometry, Molecular Weight, SPR Assay, Injection, Chromatin Immunoprecipitation

    NID-extracted DNA of both high and low copy number plasmids perform well in common downstream applications. ( A ) Restriction endonuclease digestion of the high copy number pLTM330 plasmid containing 2 SacI sites separated by approximately 400 bp. extracted by either alkali or NIDs. XL1-Blue cells harboring pLTM330 in 1.5 ml LB cultures were used for isolation. After alcohol precipitation, alkali DNA pellets were rinsed with 0.5 ml ethanol and dissolved in 40 µl TE buffer, but NID DNA pellets were directly dissolved in 40 µl TE buffer. 9 µl aliquots of native DNA of two independently isolated samples were loaded in lanes 1, 2 (alkali, A) and lanes 3, 4 (NID, N). Densitometry analysis is reported for lanes 1–4. 9 µl DNA was also used for every DNA restriction and ligation reaction in 11 µl total volume. Restriction digests with the indicated amount of restriction enzyme for the indicated length of incubation at 37°C are shown in lanes 5, 7, 9, 11 for the alkali method and lanes 6, 8, 10, 12 for the NID protocol. Arrow indicates “irreversibly denatured” DNA [1] , [25] , [29] . Arrowhead indicates the 400 bp band, and densitometry analysis of this band is reported for lanes 5–12. DNA restriction reactions were carried out in NEB1 buffer. ( B ) Plasmid samples digested using 2u SacI for 1 hour (lanes 1,4) were ligated using 0.1 Weiss unit T4 DNA ligase at 15°C for 30 minutes (lanes 2,5). Ligation products were then re-digested using 2u SacI for 1 hour (lanes 3,6). DNA restriction and ligation reactions were carried out in NEB1 buffer, but 1 mM ATP was added for the ligation reactions. Arrowhead indicates 400 bp band. B = cut sample. L = ligated sample. D = re-cut sample. Lane 2 shows the intermediate ligation products of the 400 bp DNA fragment in lane 1. ( C ) DH5α cells harboring the low copy number plasmid pEL04, containing 2 KpnI sites separated by approximately 1.5 Kb. NID plasmid isolation was performed as reported in Materials and Methods , except that lysozyme concentration in the extraction buffer was 50 µg/ml. 9 µl DNA in 11 µl total reaction volume were used for digestions. Lane 1: native DNA (N). Lane 2: DNA digestion by 1.5u KpnI. Lane 3: 3u KpnI. Lane 4: 5u KpnI. The incubation times were 1 hour. Lane 5: 1 Kb Plus DNA Ladder (Invitrogen). Densitometry analysis of the expected 1.5 Kb digestion product is reported in lanes 2–4.

    Journal: PLoS ONE

    Article Title: A One-Step Miniprep for the Isolation of Plasmid DNA and Lambda Phage Particles

    doi: 10.1371/journal.pone.0023457

    Figure Lengend Snippet: NID-extracted DNA of both high and low copy number plasmids perform well in common downstream applications. ( A ) Restriction endonuclease digestion of the high copy number pLTM330 plasmid containing 2 SacI sites separated by approximately 400 bp. extracted by either alkali or NIDs. XL1-Blue cells harboring pLTM330 in 1.5 ml LB cultures were used for isolation. After alcohol precipitation, alkali DNA pellets were rinsed with 0.5 ml ethanol and dissolved in 40 µl TE buffer, but NID DNA pellets were directly dissolved in 40 µl TE buffer. 9 µl aliquots of native DNA of two independently isolated samples were loaded in lanes 1, 2 (alkali, A) and lanes 3, 4 (NID, N). Densitometry analysis is reported for lanes 1–4. 9 µl DNA was also used for every DNA restriction and ligation reaction in 11 µl total volume. Restriction digests with the indicated amount of restriction enzyme for the indicated length of incubation at 37°C are shown in lanes 5, 7, 9, 11 for the alkali method and lanes 6, 8, 10, 12 for the NID protocol. Arrow indicates “irreversibly denatured” DNA [1] , [25] , [29] . Arrowhead indicates the 400 bp band, and densitometry analysis of this band is reported for lanes 5–12. DNA restriction reactions were carried out in NEB1 buffer. ( B ) Plasmid samples digested using 2u SacI for 1 hour (lanes 1,4) were ligated using 0.1 Weiss unit T4 DNA ligase at 15°C for 30 minutes (lanes 2,5). Ligation products were then re-digested using 2u SacI for 1 hour (lanes 3,6). DNA restriction and ligation reactions were carried out in NEB1 buffer, but 1 mM ATP was added for the ligation reactions. Arrowhead indicates 400 bp band. B = cut sample. L = ligated sample. D = re-cut sample. Lane 2 shows the intermediate ligation products of the 400 bp DNA fragment in lane 1. ( C ) DH5α cells harboring the low copy number plasmid pEL04, containing 2 KpnI sites separated by approximately 1.5 Kb. NID plasmid isolation was performed as reported in Materials and Methods , except that lysozyme concentration in the extraction buffer was 50 µg/ml. 9 µl DNA in 11 µl total reaction volume were used for digestions. Lane 1: native DNA (N). Lane 2: DNA digestion by 1.5u KpnI. Lane 3: 3u KpnI. Lane 4: 5u KpnI. The incubation times were 1 hour. Lane 5: 1 Kb Plus DNA Ladder (Invitrogen). Densitometry analysis of the expected 1.5 Kb digestion product is reported in lanes 2–4.

    Article Snippet: DH5α cells were supplied by Invitrogen (Carlsbad, CA).

    Techniques: Low Copy Number, Plasmid Preparation, Isolation, Ligation, Incubation, Concentration Assay

    Elevated temperatures and osmolytes increase the efficiency of plasmid DNA extraction by NIDs. Extractions of either pUC19 (lanes 1–8 and 10–15) or pCYPAC3 plasmids (lane 9) were carried out. Transformed DH5α cells were grown in 1.5 ml LB cultures and resuspended in 150 µl 50 mM Tris pH 8, 10 mM EDTA with or without co-solutes. 500 µg/ml lysozyme was also added, and the cells were incubated as specified below. Salt concentrations were adjusted to 0.5 M NaCl in all extracts, except the ones loaded in lanes 10 and 12–15. Extracts were cleared by centrifugation, and precipitated with 150 µl isopropanol. DNA pellets were dissolved in 40 µl TE buffer, 40 µg/ml RNase A. 10 µl aliquots of the solutions containing either the pCYPAC3 (lane 9) or pUC19 plasmids (all other lanes) were loaded on the gel. Exposure times and temperatures of extraction are shown above the lanes. For lanes 1–9, extraction with the indicated NID was done in the absence of co-solutes. For lanes 10–15, the included co-solute is indicated. Lane 1: 0.5% IGEPAL CA-630. Lane 2: 0.5% TX-100. Lane 3: 0.5% IGEPAL CA-720. Lane 4: 0.5% Tween-80. Lane 5: 0.5% Tween-20. Lane 6: 2% IGEPAL CA-720. Lane 7: 4% IGEPAL CA-720. Lane 8: 0.5% Tween 80. Lane 9: 0.5% Tween 80. Lane 10: 0.5% Tween 20/0.5 M KCl. Lane 11: 0.5% Tween 20/22.5% sucrose. Lane 12: 0.5% IGEPAL CA-630/0.5 M NH 4 Cl. Lane 13: 0.5% TX-100/0.5 M NH 4 Cl. Lane 14: 0.5% TX-100/0.5 M NaCl. Lane 15: 0.5% TX-100/0.5 M NaAc.

    Journal: PLoS ONE

    Article Title: A One-Step Miniprep for the Isolation of Plasmid DNA and Lambda Phage Particles

    doi: 10.1371/journal.pone.0023457

    Figure Lengend Snippet: Elevated temperatures and osmolytes increase the efficiency of plasmid DNA extraction by NIDs. Extractions of either pUC19 (lanes 1–8 and 10–15) or pCYPAC3 plasmids (lane 9) were carried out. Transformed DH5α cells were grown in 1.5 ml LB cultures and resuspended in 150 µl 50 mM Tris pH 8, 10 mM EDTA with or without co-solutes. 500 µg/ml lysozyme was also added, and the cells were incubated as specified below. Salt concentrations were adjusted to 0.5 M NaCl in all extracts, except the ones loaded in lanes 10 and 12–15. Extracts were cleared by centrifugation, and precipitated with 150 µl isopropanol. DNA pellets were dissolved in 40 µl TE buffer, 40 µg/ml RNase A. 10 µl aliquots of the solutions containing either the pCYPAC3 (lane 9) or pUC19 plasmids (all other lanes) were loaded on the gel. Exposure times and temperatures of extraction are shown above the lanes. For lanes 1–9, extraction with the indicated NID was done in the absence of co-solutes. For lanes 10–15, the included co-solute is indicated. Lane 1: 0.5% IGEPAL CA-630. Lane 2: 0.5% TX-100. Lane 3: 0.5% IGEPAL CA-720. Lane 4: 0.5% Tween-80. Lane 5: 0.5% Tween-20. Lane 6: 2% IGEPAL CA-720. Lane 7: 4% IGEPAL CA-720. Lane 8: 0.5% Tween 80. Lane 9: 0.5% Tween 80. Lane 10: 0.5% Tween 20/0.5 M KCl. Lane 11: 0.5% Tween 20/22.5% sucrose. Lane 12: 0.5% IGEPAL CA-630/0.5 M NH 4 Cl. Lane 13: 0.5% TX-100/0.5 M NH 4 Cl. Lane 14: 0.5% TX-100/0.5 M NaCl. Lane 15: 0.5% TX-100/0.5 M NaAc.

    Article Snippet: DH5α cells were supplied by Invitrogen (Carlsbad, CA).

    Techniques: Plasmid Preparation, DNA Extraction, Transformation Assay, Incubation, Centrifugation

    Comparison of bioluminescence from S. aureus and E. coli containing the native luxCDABE operon versus that in strains containing the modified luxABCDE operon. Exponential cultures of S. aureus RN4220 pMK4 luxABCDE P1 (-■-), S. aureus RN4220 pMK4 luxCDABE P1 (-▴-), E. coli DH5α pMK4 luxABCDE P1 (‥■‥) and E. coli DH5α pMK4 luxCDABE P1 (‥▴‥) were diluted across black 96-well microtiter plates in doubling dilutions (−0.3 log) and monitored for light over a period of 30 min using the ICCD camera. The content of each well was then plated to allow the number of CFU to be compared to the level of bioluminescence (RLU).

    Journal: Infection and Immunity

    Article Title: Monitoring Bioluminescent Staphylococcus aureus Infections in Living Mice Using a Novel luxABCDE Construct

    doi:

    Figure Lengend Snippet: Comparison of bioluminescence from S. aureus and E. coli containing the native luxCDABE operon versus that in strains containing the modified luxABCDE operon. Exponential cultures of S. aureus RN4220 pMK4 luxABCDE P1 (-■-), S. aureus RN4220 pMK4 luxCDABE P1 (-▴-), E. coli DH5α pMK4 luxABCDE P1 (‥■‥) and E. coli DH5α pMK4 luxCDABE P1 (‥▴‥) were diluted across black 96-well microtiter plates in doubling dilutions (−0.3 log) and monitored for light over a period of 30 min using the ICCD camera. The content of each well was then plated to allow the number of CFU to be compared to the level of bioluminescence (RLU).

    Article Snippet: This ligation was again electroporated into E. coli DH5α, and luxAB clones and selected by screening for bioluminescence in the presence of exogenously added n -decyl aldehyde (Sigma, St. Louis, Mo.).

    Techniques: Modification

    Temperature stability of the modified luxABCDE operon. Exponential cultures of S. aureus RN4220 pMK4 luxABCDE P1 (-■-), E. coli DH5α pMK4 luxABCDE P1 (‥■‥), and E. coli DH5α pMK4 luxCDABE P1 (‥▴‥) were grown to approximately 1 × 10 7 CFU/ml at 30°C and 1-ml volumes of each were placed in heating blocks set at 31, 33, 35, 37, 39, 41, 43, 45, and 47°C. After 1 h at each of these elevated temperatures, the nine heating blocks were sequentially placed inside a dark chamber and light from each of the three cultures was recorded for a period of 1 min using the ICCD camera. Shown are the number of RLU at each of the temperatures, with this data expressed as a percentage of the maximum (Max) bioluminescence attained and adjusted for variation in the number of CFU.

    Journal: Infection and Immunity

    Article Title: Monitoring Bioluminescent Staphylococcus aureus Infections in Living Mice Using a Novel luxABCDE Construct

    doi:

    Figure Lengend Snippet: Temperature stability of the modified luxABCDE operon. Exponential cultures of S. aureus RN4220 pMK4 luxABCDE P1 (-■-), E. coli DH5α pMK4 luxABCDE P1 (‥■‥), and E. coli DH5α pMK4 luxCDABE P1 (‥▴‥) were grown to approximately 1 × 10 7 CFU/ml at 30°C and 1-ml volumes of each were placed in heating blocks set at 31, 33, 35, 37, 39, 41, 43, 45, and 47°C. After 1 h at each of these elevated temperatures, the nine heating blocks were sequentially placed inside a dark chamber and light from each of the three cultures was recorded for a period of 1 min using the ICCD camera. Shown are the number of RLU at each of the temperatures, with this data expressed as a percentage of the maximum (Max) bioluminescence attained and adjusted for variation in the number of CFU.

    Article Snippet: This ligation was again electroporated into E. coli DH5α, and luxAB clones and selected by screening for bioluminescence in the presence of exogenously added n -decyl aldehyde (Sigma, St. Louis, Mo.).

    Techniques: Modification

    Immunoprecipitation to determine the specificity of DivIVA interactions. (A) Immunoprecipitation of GFP-DivIVA-cI-Spo0J complex (lane 1), GFP-Spo0J-cI-DivIVA complex (lane 2), GFP-FtsZ-cI-DivIVA complex (lane 3), and GFP-DivIVA-cI-FtsZ complex (lane 4). (B) Immunoprecipitation of cI-DivIVA-GFP-PrfA complex (lane 1). Protein extracts were immunoprecipitated with anti-GFP antibodies and were detected with anti-cI rabbit polyclonal antibodies by Western blotting as described in Materials and Methods. Lane M contained molecular weight markers. In panel B lane 2 contained crude extract of E. coli strain DH5α expressing cI-DivIVA, detected with anti-cI antibodies.

    Journal: Journal of Bacteriology

    Article Title: Streptococcus pneumoniae DivIVA: Localization and Interactions in a MinCD-Free Context ▿ DivIVA: Localization and Interactions in a MinCD-Free Context ▿ †

    doi: 10.1128/JB.01168-06

    Figure Lengend Snippet: Immunoprecipitation to determine the specificity of DivIVA interactions. (A) Immunoprecipitation of GFP-DivIVA-cI-Spo0J complex (lane 1), GFP-Spo0J-cI-DivIVA complex (lane 2), GFP-FtsZ-cI-DivIVA complex (lane 3), and GFP-DivIVA-cI-FtsZ complex (lane 4). (B) Immunoprecipitation of cI-DivIVA-GFP-PrfA complex (lane 1). Protein extracts were immunoprecipitated with anti-GFP antibodies and were detected with anti-cI rabbit polyclonal antibodies by Western blotting as described in Materials and Methods. Lane M contained molecular weight markers. In panel B lane 2 contained crude extract of E. coli strain DH5α expressing cI-DivIVA, detected with anti-cI antibodies.

    Article Snippet: The ligation mixture was transformed into E. coli DH5α competent cells (Invitrogen), and transformants were selected after overnight growth on LB medium plates with ampicillin.

    Techniques: Immunoprecipitation, Western Blot, Molecular Weight, Expressing

    YegI is an integral membrane protein A) Predicted topology of YegI using Phobius. Amino acid positions at which phoA-lacZα reporters are fused are indicated along with associated phenotypes. Phenotypes of phoAlacZα fusions due to LacZ activity are denoted as red ellipses while PhoA activity are denoted as blue ellipses. Predicted transmembrane domains (I and II) are depicted as grey rectangles. (B) Membrane topology analysis: DH5α transformed with different phoAlacZα reporters were streaked on an LB plate containing 5-bromo-4-chloro-3-indolyl phosphate disodium salt (X-Phos, 80μg/ml), 6-chloro-3-indolyl-β-D-galactoside (Red-Gal, 100 μg/ml, 0.1 % w/v arabinose and 100 μg/ml of ampicillin. Control pBAD24: control strain expressing pBAD24 empty vector, control phoAlacZα: strain expressing phoAlacZα in pBAD24 without YegI. Amino acid positions at which phoAlacZα reporters are fused are indicated.

    Journal: bioRxiv

    Article Title: Identification and biochemical characterization of a novel eukaryotic-like Ser/Thr kinase in E. coli

    doi: 10.1101/819920

    Figure Lengend Snippet: YegI is an integral membrane protein A) Predicted topology of YegI using Phobius. Amino acid positions at which phoA-lacZα reporters are fused are indicated along with associated phenotypes. Phenotypes of phoAlacZα fusions due to LacZ activity are denoted as red ellipses while PhoA activity are denoted as blue ellipses. Predicted transmembrane domains (I and II) are depicted as grey rectangles. (B) Membrane topology analysis: DH5α transformed with different phoAlacZα reporters were streaked on an LB plate containing 5-bromo-4-chloro-3-indolyl phosphate disodium salt (X-Phos, 80μg/ml), 6-chloro-3-indolyl-β-D-galactoside (Red-Gal, 100 μg/ml, 0.1 % w/v arabinose and 100 μg/ml of ampicillin. Control pBAD24: control strain expressing pBAD24 empty vector, control phoAlacZα: strain expressing phoAlacZα in pBAD24 without YegI. Amino acid positions at which phoAlacZα reporters are fused are indicated.

    Article Snippet: Ligation products were transformed in DH5α cells and selected on LB/ampicillin plates.

    Techniques: Activity Assay, Transformation Assay, Expressing, Plasmid Preparation

    Strategy for gene replacement in Streptomyces . ( A )IV (Apra R ), oriT of RK2, and two FRT sites was amplified by PCR by using primers containing 39-nt 5′ homology extensions corresponding to regions flanking the S. coelicolor target gene (dotted lines a and b) and 20-nt 3′ homology to the unique priming sites P1 and P2 of the disruption cassette. ( B ) The PCR product from A was used to transform E. coli BW25113/pIJ790 (expressing the λ-Red recombination functions), containing the Supercos-1 cosmid with the cloned target gene ( cyc2 ). ( C ) Apra R transformants were selected, and the recombinant cosmid was identified by PCR and restriction analysis. ( D ) The potent methyl-specific restriction system of S. coelicolor was circumvented by introducing the recombinant cosmid into the methylation-deficient E. coli host ET12567/pUZ8002. The cosmid was mobilized in trans by pUZ8002 to Streptomyces by conjugation. ( E ) Selected exconjugants (Apra R ) were screened for Kan S (loss of the Kan resistance gene neo ), and the double cross-over allelic exchange was confirmed by PCR and Southern blot analysis. ( F ) E. coli DH5α cells containing pCP20 were transformed with the mutagenized cosmid DNA, and FLP synthesis was induced. ( G) The loss of the disruption resistance marker was tested, and the cosmid was introduced by polyethylene glycol (PEG)-mediated transformation into the S. coelicolor mutant carrying a marked deletion within the gene of interest. ( H ) Kan R transformants arising from single cross-over events were restreaked nonselectively and screened for the loss of both Kan R and Apra R , indicating a successful replacement by the in-frame deletion marked only by a “scar” sequence of 81 bp containing priming sites P1 and P2 (underlined), and one FRT site.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin

    doi: 10.1073/pnas.0337542100

    Figure Lengend Snippet: Strategy for gene replacement in Streptomyces . ( A )IV (Apra R ), oriT of RK2, and two FRT sites was amplified by PCR by using primers containing 39-nt 5′ homology extensions corresponding to regions flanking the S. coelicolor target gene (dotted lines a and b) and 20-nt 3′ homology to the unique priming sites P1 and P2 of the disruption cassette. ( B ) The PCR product from A was used to transform E. coli BW25113/pIJ790 (expressing the λ-Red recombination functions), containing the Supercos-1 cosmid with the cloned target gene ( cyc2 ). ( C ) Apra R transformants were selected, and the recombinant cosmid was identified by PCR and restriction analysis. ( D ) The potent methyl-specific restriction system of S. coelicolor was circumvented by introducing the recombinant cosmid into the methylation-deficient E. coli host ET12567/pUZ8002. The cosmid was mobilized in trans by pUZ8002 to Streptomyces by conjugation. ( E ) Selected exconjugants (Apra R ) were screened for Kan S (loss of the Kan resistance gene neo ), and the double cross-over allelic exchange was confirmed by PCR and Southern blot analysis. ( F ) E. coli DH5α cells containing pCP20 were transformed with the mutagenized cosmid DNA, and FLP synthesis was induced. ( G) The loss of the disruption resistance marker was tested, and the cosmid was introduced by polyethylene glycol (PEG)-mediated transformation into the S. coelicolor mutant carrying a marked deletion within the gene of interest. ( H ) Kan R transformants arising from single cross-over events were restreaked nonselectively and screened for the loss of both Kan R and Apra R , indicating a successful replacement by the in-frame deletion marked only by a “scar” sequence of 81 bp containing priming sites P1 and P2 (underlined), and one FRT site.

    Article Snippet: E. coli DH5α (Stratagene) was used as a host for plasmid constructions.

    Techniques: Amplification, Polymerase Chain Reaction, Expressing, Clone Assay, Recombinant, Methylation, Conjugation Assay, Southern Blot, Transformation Assay, Cosmid DNA, Marker, Mutagenesis, Sequencing

    D1 anaerobic protein purity. Protein was expressed in DH5α from the pASG-IBA35 vector A. Steps of D1 anaerobic purification process. Numbers above the gel mark fractions: 1 – before induction, 2- after induction, 3 – lysate, 4- pellet after centrifugation at 40 000 rcf, 5- NiNTA flow through, 6 – elution (this D1 anaerobic protein batch was used for EXAFS experiments). The marked band with mass 30 kDa corresponds to D1 protein. B. The purity of D1 anaerobic protein batch used for TSA, crystallization and SEC HPLC analysis (lanes 1 and 2).

    Journal: Protein expression and purification

    Article Title: Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization

    doi: 10.1016/j.pep.2017.03.019

    Figure Lengend Snippet: D1 anaerobic protein purity. Protein was expressed in DH5α from the pASG-IBA35 vector A. Steps of D1 anaerobic purification process. Numbers above the gel mark fractions: 1 – before induction, 2- after induction, 3 – lysate, 4- pellet after centrifugation at 40 000 rcf, 5- NiNTA flow through, 6 – elution (this D1 anaerobic protein batch was used for EXAFS experiments). The marked band with mass 30 kDa corresponds to D1 protein. B. The purity of D1 anaerobic protein batch used for TSA, crystallization and SEC HPLC analysis (lanes 1 and 2).

    Article Snippet: During the course of the study two E. coli strains, DH5α (#18265017, LifeTechnologies) and BL21(DE3)RILP (#230280, Agilent Technologies) were used.

    Techniques: Plasmid Preparation, Purification, Centrifugation, Flow Cytometry, Crystallization Assay, Size-exclusion Chromatography, High Performance Liquid Chromatography

    H. pylori produces a luxS -dependent AI-2 activity. Cell-free supernatants were tested for the ability to induce luminescence expression in V. harveyi BB170. Ten percent cell-free supernatants or sterile media were mixed with the reporter strain in microtiter plates and incubated at 30°C on a rotary shaker. Aliquots were taken, and both cell density and light production were determined. Activity is reported as fold activation of luminescence of BB170 over the level of luminescence when sterile media were added. Assays were repeated at least two times. (A) Wild-type H. pylori and Typhimurium 14028 supernatants contain signaling substances that induce expression of luminescence in V. harveyi BB170, while the luxS deletion strain EJ103 does not. (B) H. pylori luxS expression in E. coli DH5α restores AI-2 activity. The error bars indicate standard deviations.

    Journal: Journal of Bacteriology

    Article Title: Evidence for a Signaling System in Helicobacter pylori: Detection of a luxS-Encoded Autoinducer

    doi:

    Figure Lengend Snippet: H. pylori produces a luxS -dependent AI-2 activity. Cell-free supernatants were tested for the ability to induce luminescence expression in V. harveyi BB170. Ten percent cell-free supernatants or sterile media were mixed with the reporter strain in microtiter plates and incubated at 30°C on a rotary shaker. Aliquots were taken, and both cell density and light production were determined. Activity is reported as fold activation of luminescence of BB170 over the level of luminescence when sterile media were added. Assays were repeated at least two times. (A) Wild-type H. pylori and Typhimurium 14028 supernatants contain signaling substances that induce expression of luminescence in V. harveyi BB170, while the luxS deletion strain EJ103 does not. (B) H. pylori luxS expression in E. coli DH5α restores AI-2 activity. The error bars indicate standard deviations.

    Article Snippet: The bacterial strains used were the clinical isolate H. pylori Alston, a generous gift from David Cave (St. Elizabeth's Hospital, Boston, Mass.), V. harveyi BB170 , and E. coli DH5α (Bio-Rad Laboratories).

    Techniques: Activity Assay, Expressing, Incubation, Activation Assay

    Dynamic monitoring of intestinal epithelial cell responses to intestinal bacteria. HT-29 cells (10,000 cells per well) were seeded into E-plates. The next day, cells were infected with Y. enterocolitica (A), S. flexneri (B), L. monocytogenes (C), E. coli DH5α (E), and Lactobacillus (F). Arrows, bacterial addition. Representative curves are an average of four replicate wells.

    Journal: PLoS ONE

    Article Title: Phenotypic Pattern-Based Assay for Dynamically Monitoring Host Cellular Responses to Salmonella Infections

    doi: 10.1371/journal.pone.0026544

    Figure Lengend Snippet: Dynamic monitoring of intestinal epithelial cell responses to intestinal bacteria. HT-29 cells (10,000 cells per well) were seeded into E-plates. The next day, cells were infected with Y. enterocolitica (A), S. flexneri (B), L. monocytogenes (C), E. coli DH5α (E), and Lactobacillus (F). Arrows, bacterial addition. Representative curves are an average of four replicate wells.

    Article Snippet: E. coli DH5α was from Tiangen Biotech (Beijing, China).

    Techniques: Infection

    Efficiency of electroporation of pTOPO/FNL10 and pFNLTP1 into E. coli DH5α or Francisella spp. ( F. tularensis LVS or F. novicida U112). The transformation efficiency, expressed as log CFU per microgram of DNA (mean ± standard deviation), was determined from three individual preparations of competent cells. Competent cells of E. coli were prepared with 10% glycerol, while 0.5 M sucrose was used to prepare competent cells of F. tularensis LVS and F. novicida U112. Most electroporations were performed with 100 ng of plasmid DNA; the only exception was the electroporation of pTOPO/FNL10 into F. tularensis LVS, for which 500 ng was used. The strains indicated in parentheses are the hosts from which plasmid DNA was isolated.

    Journal: Applied and Environmental Microbiology

    Article Title: Construction and Characterization of a Highly Efficient Francisella Shuttle Plasmid

    doi: 10.1128/AEM.70.12.7511-7519.2004

    Figure Lengend Snippet: Efficiency of electroporation of pTOPO/FNL10 and pFNLTP1 into E. coli DH5α or Francisella spp. ( F. tularensis LVS or F. novicida U112). The transformation efficiency, expressed as log CFU per microgram of DNA (mean ± standard deviation), was determined from three individual preparations of competent cells. Competent cells of E. coli were prepared with 10% glycerol, while 0.5 M sucrose was used to prepare competent cells of F. tularensis LVS and F. novicida U112. Most electroporations were performed with 100 ng of plasmid DNA; the only exception was the electroporation of pTOPO/FNL10 into F. tularensis LVS, for which 500 ng was used. The strains indicated in parentheses are the hosts from which plasmid DNA was isolated.

    Article Snippet: E. coli DH5α and HB101 were grown at 37°C aerobically in Luria-Bertani (LB) medium (Difco) supplemented with kanamycin (50 μg ml−1 ), streptomycin (100 μg ml−1 ), or ampicillin (100 μg ml−1 ) when required.

    Techniques: Electroporation, Transformation Assay, Standard Deviation, Plasmid Preparation, Isolation