Journal:

Article Title: The p300/CBP acetyltransferases function as transcriptional coactivators of ?-catenin in vertebrates

doi: 10.1093/emboj/19.8.1839

Figure Lengend Snippet: Fig. 1. β-catenin utilizes p300 as coactivator at the siamois promoter. (A) Schematic representation of β-catenin and p300. In β-catenin (hatched bar) the Armadillo repeats (gray boxes) and interaction domains for various binding partners are marked. The GSK-3β target sequence (GSK) and the transactivating elements (N-TAs, C-TAs) are also shown. Autonomous TAs: dark gray; auxiliary TAs: light gray. In β-catS33A serine/threonine residues at positions 33, 37, 41 and 45 in the GSK-3β target sequence were substituted with alanines. In p300 the cysteine/histidine-rich elements CH-1, CH-2, and CH-3, the Bromo domain (gray boxes), the HAT domain (black bar) and regions interacting with nuclear hormone receptors (NHR), other transcription factors and E1A are denoted. (B) 293 cells were transfected with luciferase reporter plasmids harboring either the WT siamois promoter or a deletion mutant lacking the TCF binding sites (1.0 µg), the lacZ expression plasmid pCMVβ (Promega) (0.5 µg), a construct for expression of β-catS33A (0.5 µg) and increasing amounts of expression vector for p300 (0.5, 1.0, 2.0 µg) as indicated. Luciferase activities were determined 40 h post transfection, normalized against β-galactosidase values and expressed as relative activity compared with cells transfected with the WT siamois reporter alone (rel. activity: 1). (C) 293 cells were transfected with the WT siamois reporter, pCMVβ and expression vectors for β-catS33A (0.5 µg), p300 (2.0 µg) and WT E1A or mutant E1A (0.2 µg) as indicated. Reporter activity without β-catenin and p300: 1. A diagram of 12S-E1A is depicted. Conserved regions 1 and 2 (CR1, CR2), domains involved in binding Rb-family members, p300/CBP and the CtBP transcriptional repressor, and the location of the mRb and mCBP mutations are indicated. (B, C) Average values from at least three independent experiments are shown with standard deviations. (D) SW480 cells were transfected with expression vectors (2.5 µg each) for the E1A proteins as indicated. Whole cell lysates were prepared 40 h after transfection and E1A expression levels were analyzed by Western blotting with a monoclonal antibody. Mw: molecular weight markers.

Article Snippet: In p300 the cysteine/histidine-rich elements CH-1, CH-2, and CH-3, the Bromo domain (gray boxes), the HAT domain (black bar) and regions interacting with nuclear hormone receptors (NHR), other transcription factors and E1A are denoted. ( B ) 293 cells were transfected with luciferase reporter plasmids harboring either the WT siamois promoter or a deletion mutant lacking the TCF binding sites (1.0 μg), the lacZ expression plasmid pCMVβ (Promega) (0.5 μg), a construct for expression of β-catS33A (0.5 μg) and increasing amounts of expression vector for p300 (0.5, 1.0, 2.0 μg) as indicated.

Techniques: Binding Assay, Sequencing, Transfection, Luciferase, Mutagenesis, Expressing, Plasmid Preparation, Construct, Activity Assay, Western Blot, Molecular Weight

Journal:

Article Title: The p300/CBP acetyltransferases function as transcriptional coactivators of ?-catenin in vertebrates

doi: 10.1093/emboj/19.8.1839

Figure Lengend Snippet: Fig. 2. Promoter-specific differences in transcriptional activation mediated by β-catenin and p300. (A) 293 cells were transfected with the TOPFLASH or FOPFLASH reporter plasmids (1.0 µg), pCMVβ (0.5 µg), a construct for expression of β-catS33A (0.5 µg) and increasing amounts of expression vector for p300 (0.5, 1.0, 2.0 µg) as indicated. Luciferase activities were determined as before. (B) Same experiment as in (A) but with a reporter construct harboring the cyclin D1 promoter. Reporter activity in (A) and (B) without β-catenin and p300: 1.

Article Snippet: In p300 the cysteine/histidine-rich elements CH-1, CH-2, and CH-3, the Bromo domain (gray boxes), the HAT domain (black bar) and regions interacting with nuclear hormone receptors (NHR), other transcription factors and E1A are denoted. ( B ) 293 cells were transfected with luciferase reporter plasmids harboring either the WT siamois promoter or a deletion mutant lacking the TCF binding sites (1.0 μg), the lacZ expression plasmid pCMVβ (Promega) (0.5 μg), a construct for expression of β-catS33A (0.5 μg) and increasing amounts of expression vector for p300 (0.5, 1.0, 2.0 μg) as indicated.

Techniques: Activation Assay, Transfection, Construct, Expressing, Plasmid Preparation, Luciferase, Activity Assay

Journal:

Article Title: The p300/CBP acetyltransferases function as transcriptional coactivators of ?-catenin in vertebrates

doi: 10.1093/emboj/19.8.1839

Figure Lengend Snippet: Fig. 3. (A) Endogenous β-catenin in SW480 cells utilizes p300 as coactivator. SW480 cells were transfected with pCMVβ (0.5 µg), the TOPFLASH/FOPFLASH (1.5 µg) or siamois promoter constructs (2.5 µg) and expression vectors for p300 (2.0 µg) or dominant negative ΔN-TCF4 (2.0 µg) as indicated. To interfere with p300-mediated transcriptional activation WT E1A, E1AmRb, E1AmCBP or E1AΔCR1 were cotransfected (0.2 µg each). Luciferase activities were determined as before. Activity of the TOPFLASH or WT siamois reporter without exogenous p300, ΔN-TCF4 or E1A was set as 100% (n.d.: not done). (B) Functional similarity of p300 and CBP. SW480 cells were transfected with pCMVβ (0.5 µg), the TOPFLASH/FOPFLASH reporter constructs (1.5 µg) and expression vectors for p300 (4.0 µg) or CBP (1.0, 2.0 and 4.0 µg) as indicated. Luciferase activities, determined as before, are expressed as relative activities compared with cells transfected with FOPFLASH alone. (C) HAT activity of p300 is not required for its function as coactivator of β-catenin. 293 cells were transfected with the siamois reporter (0.5 µg), pCMVβ (0.1 µg), and expression vectors for β-catS33A (0.5 µg), WT p300, or HAT-defective p300mut (0.5 and 2.5 µg) as indicated. Control transfectants received 2.5 µg of empty expression vector or p300 plasmids. Luciferase activities were determined as before. Reporter activity without β-catenin and p300: 1.

Article Snippet: In p300 the cysteine/histidine-rich elements CH-1, CH-2, and CH-3, the Bromo domain (gray boxes), the HAT domain (black bar) and regions interacting with nuclear hormone receptors (NHR), other transcription factors and E1A are denoted. ( B ) 293 cells were transfected with luciferase reporter plasmids harboring either the WT siamois promoter or a deletion mutant lacking the TCF binding sites (1.0 μg), the lacZ expression plasmid pCMVβ (Promega) (0.5 μg), a construct for expression of β-catS33A (0.5 μg) and increasing amounts of expression vector for p300 (0.5, 1.0, 2.0 μg) as indicated.

Techniques: Transfection, Construct, Expressing, Dominant Negative Mutation, Activation Assay, Luciferase, Activity Assay, Functional Assay, Control, Plasmid Preparation

Journal:

Article Title: The p300/CBP acetyltransferases function as transcriptional coactivators of ?-catenin in vertebrates

doi: 10.1093/emboj/19.8.1839

Figure Lengend Snippet: Fig. 5. Interactions between p300 and β-catenin in vivo. (A) Co-immunoprecipitation of p300 and β-catenin. 293 cells were transfected with expression plasmids for HA-tagged p300 and myc-tagged β-catS33A as indicated. Cell extracts were prepared 40 h after transfection and used for immunoprecipitations with anti-HA or anti-myc antibodies. Immunoprecipitated material and a fraction of each lysate was resolved by SDS–PAGE and analyzed by Western blotting with antibodies as shown. IP: immunoprecipitation; IB: immunoblot. (B) Mammalian two-hybrid analyses of interactions between β-catenin and p300. NIH 3T3 cells were transfected with pCMVβ (0.05 µg), the Gal4-dependent pG5E1bLuc reporter construct (0.1 µg) and empty control vectors or expression vectors for Gal4–p300 (1737–1891) (0.5 µg), LEFΔN–VP16 (0.5 µg) and β-catenin–VP16 fusions (0.5 µg) as indicated. Luciferase activities, determined as before, are expressed as relative activity compared with cells transfected with the pG5E1bLuc reporter and empty expression vector (rel. activity: 1). The diagram on the right shows the structure of the β-catenin–VP16 and the LEFΔN–VP16 prey fusions, the Gal4–p300 (1737–1891) bait and the pG5E1bLuc reporter. In β-catenin, the GSK recognition sequence, N-terminal and C-terminal transactivating elements (N-TAs, C-TAs) and the p300 interaction domain (black bar) are marked. LEFΔN–VP16 contains the HMG-box (hatched box) but lacks the β-catenin binding site. The Gal4–p300 fusion harbors the C-terminal portion of the CH-3 domain (dark gray box). VP16: VP16 transactivation domain.

Article Snippet: In p300 the cysteine/histidine-rich elements CH-1, CH-2, and CH-3, the Bromo domain (gray boxes), the HAT domain (black bar) and regions interacting with nuclear hormone receptors (NHR), other transcription factors and E1A are denoted. ( B ) 293 cells were transfected with luciferase reporter plasmids harboring either the WT siamois promoter or a deletion mutant lacking the TCF binding sites (1.0 μg), the lacZ expression plasmid pCMVβ (Promega) (0.5 μg), a construct for expression of β-catS33A (0.5 μg) and increasing amounts of expression vector for p300 (0.5, 1.0, 2.0 μg) as indicated.

Techniques: In Vivo, Immunoprecipitation, Transfection, Expressing, SDS Page, Western Blot, Construct, Control, Luciferase, Activity Assay, Plasmid Preparation, Sequencing, Binding Assay

Journal: PLoS ONE

Article Title: Green Tea Polyphenols Induce p53-Dependent and p53-Independent Apoptosis in Prostate Cancer Cells through Two Distinct Mechanisms

doi: 10.1371/journal.pone.0052572

Figure Lengend Snippet: Isogenic cells with lenti-virus vector background were generated by permanent knockdown of p53 in LNCaP cells [LNCaPshp53] and transfected with control vector, LNCaPshV cells. [ A ] Cell treated with 50 and 100 ng/ml camptothecin for 24 h were harvested, stained with PI and analyzed by flow cytometry to measure sub-G1 and G1 population. [ B ] Cells were treated with 50, 100 and 200 ng/ml of camptothecin for 24 h and stained with methylene blue. The intensity of methylene blue taken up by live cells was measured spectrophotometerically after eluting the dye in 0.1N HCl and compared with untreated cells. [ C ] Knockdown of p53 upregulated Akt/BAD signaling in prostate cancer cells. LNCaPshV and LNCaPshp53 cells were lysed and Western blotting was performed for p53, Akt, p-Akt (Ser473), BAD, p-BAD (Ser136) proteins. Actin was used as internal loading control. [ D ] Relative intensities of p-Akt and p-Bad protein in LNCaPshV and LNCaPshp53RNA cells where bands were normalized to actin and expressed in relative values compared to the native protein. The details are described in the materials and methods section.

Article Snippet: Antibodies for anti-caspase-9 (#9502), anti-caspase-3 (#9662), cleaved PARP (#9544), anti-c-IAP (#3130), anti-X-IAP (#2045), anti-SAPK/JNK (#9258), anti-p-JNK Thr183/Tyr185 (#9251), anti-p-Akt Ser473 (#4051), anti-BAD (#9292), anti-p-BAD Ser136 (#9295) and anti-VDAC (#4866) were purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Virus, Plasmid Preparation, Generated, Knockdown, Transfection, Control, Staining, Flow Cytometry, Western Blot

Journal: PLoS ONE

Article Title: Green Tea Polyphenols Induce p53-Dependent and p53-Independent Apoptosis in Prostate Cancer Cells through Two Distinct Mechanisms

doi: 10.1371/journal.pone.0052572

Figure Lengend Snippet: [ A ] Cells were treated with 20–80 µg/ml concentration of GTP for 24 h and Western blotting was performed for p53, Akt, p-Akt (Ser473), BAD, p-BAD (Ser136), p21/waf1, PUMA and Bax proteins. [ B ] Cells were treated with 40 µg/ml concentration of GTP for 3, 6, 9, 12, 24 and 48 h and Western blotting was performed for procaspase-9, cleaved caspase-9 and cleaved caspase-3 proteins. A typical actin blot demonstrates internal loading control. Cytochrome c release from mitochondria to cytosol was determined by Western blotting in GTP treated cells. A typical actin blot demonstrates internal loading control for cytosol whereas VDAC as internal loading control for mitochondria. [ D ] Cells were treated with 20 µM concentration of PI3K-Akt inhibitor LY294002 for 8 h and with 40 µg/ml GTP for 16 h alone, or LY294002 for 8 h followed by GTP treatment in combination followed by Western blotting for p-Akt, Akt, p-BAD and BAD proteins. The expressions of native proteins were considered as loading controls. [ D ] Cell death measurement was performed by photometric enzyme immunoassay Cell Death Detection ELISA kit. The bars represent mean±SD of at least two independent experiments each performed in duplicate, ** p <0.001 represents significant differences as compared to control group. [ E ] Light microscopic images of LNCaPshV and LNCaPshp53 cells treated with LY294002 and GTP alone or in combination. The details are described in the materials and methods section.

Article Snippet: Antibodies for anti-caspase-9 (#9502), anti-caspase-3 (#9662), cleaved PARP (#9544), anti-c-IAP (#3130), anti-X-IAP (#2045), anti-SAPK/JNK (#9258), anti-p-JNK Thr183/Tyr185 (#9251), anti-p-Akt Ser473 (#4051), anti-BAD (#9292), anti-p-BAD Ser136 (#9295) and anti-VDAC (#4866) were purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Concentration Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Biocatalytic Formation of Novel Polyesters with para -Hydroxyphenyl groups in the Backbone – Engineering Cupriavidus necator for production of high-performance materials from CO 2 and electricity

doi: 10.1101/2021.12.12.472320

Figure Lengend Snippet: 1 H-NMR spectra of P (3HB- co -4HB) produced by C. necator H16 Δ phaC1 pCM66T_P BAD - pct540 - phaC1437 (A) as well as P (3HB- co -4HB- co -6HC) (B), P (3HB- co -2H4PheB) (C), P (3HB- co -PheLA) (D), P (3HB- co -MA) (E), and P (3HB- co -PA) (F) produced by C. necator H16 Δ phaC1 pCM66T_P BAD - hadA - phaC1437 . Areas are indicative of the ratios for 3HB to non-natural repeat units. (A): In addition to the P3HB sextet resonance at 5.25 ppm, a triplet resonance at 4.11 ppm shows that the C4-methylene of the 4HB repeat is present, indicating incorporation of 4HB into the polymer. The large fraction of P4HB causes multiple P3HB peaks because of different possible orders in the polymer (e.g., -3HB-P3HB-P4HB-, -P4HB-P3HB-P4HB-, -P4HB-P3HB-P3HB-, -P3HB-P4HB-P3HB-). (B): In addition to the P3HB sextet resonance at 5.25 ppm and P4HB triplet resonance at 4.11 ppm, a triplet resonance at 4.07 ppm shows that the C6-methylene of the 6HC repeat is present, indicating incorporation of 6HC into the polymer. (C): In addition to the P3HB sextet resonance at 5.25 ppm, a triplet resonance at 5.13 ppm shows that the C2-methine of the 2H4PheB repeat is present. The broad aromatic multiplet resonance at 7.21 ppm is also indicative of P2H4PheB. (D): The P3HB sextet resonance at 5.25 ppm overlaps with the methine PPheLA resonance, so the methylene doublet resonances of 3.15 & 3.04 ppm and 2.58 & 2.48 ppm were used to show the presence of P3HB and PPheLA respectively. The broad aromatic resonance at 7.21 ppm is also indicative of PPheLA. (E): In addition to the P3HB sextet resonance at 5.25 ppm, a multiplet resonance at 5.12 ppm shows that the C2-methine of the MA repeat is present. The broad aromatic multiplet resonance at 7.28 ppm is also indicative of MA. (F): In addition to the P3HB sextet resonance at 5.25 ppm, two doublet resonances at 7.19 and 6.98 ppm show that the aromatic PA repeat is present, indicating incorporation of the para -hydroxyphenyl unit into the polymer.

Article Snippet: The plasmids pCM66T_P BAD - pct540 - phaC1437 and pCM66T_P BAD - hadA - phaC1437 were constructed by GenScript, cloning the synthetic operons containing pct540 & phaC1437 and hadA & phaC1437 , respectively, into pCM66T_P BAD -RFP (cf. supplementary material 2).

Techniques: Produced, Polymer

Journal: bioRxiv

Article Title: Biocatalytic Formation of Novel Polyesters with para -Hydroxyphenyl groups in the Backbone – Engineering Cupriavidus necator for production of high-performance materials from CO 2 and electricity

doi: 10.1101/2021.12.12.472320

Figure Lengend Snippet: Molecular weight distribution (M n and M W ) for PHA obtained from wild-type C. necator H16, and engineered C. necator H16 (Δ phaC1 pCM66T_P BAD - hadA - phaC1437 , non-induced and fully induced, respectively).

Article Snippet: The plasmids pCM66T_P BAD - pct540 - phaC1437 and pCM66T_P BAD - hadA - phaC1437 were constructed by GenScript, cloning the synthetic operons containing pct540 & phaC1437 and hadA & phaC1437 , respectively, into pCM66T_P BAD -RFP (cf. supplementary material 2).

Techniques: Molecular Weight

Journal: bioRxiv

Article Title: Biocatalytic Formation of Novel Polyesters with para -Hydroxyphenyl groups in the Backbone – Engineering Cupriavidus necator for production of high-performance materials from CO 2 and electricity

doi: 10.1101/2021.12.12.472320

Figure Lengend Snippet: Cultivation-profile (growth by means of OD) of representative experiment with transgenic C. necator H16 Δ phaC1 pCM66T_P BAD - hadA - phaC1437 in bio-electrochemical system (BES). Induction of expression, addition of aromatic polymer-precursor and depletion of nitrogen source as indicated. The insert on the bottom right shows a schematic of the BES, which evolves H 2 at the cathode and O 2 at the anode as electron carrier and acceptor for autotrophic growth and production of aromatic polyesters from CO 2 and electricity.

Article Snippet: The plasmids pCM66T_P BAD - pct540 - phaC1437 and pCM66T_P BAD - hadA - phaC1437 were constructed by GenScript, cloning the synthetic operons containing pct540 & phaC1437 and hadA & phaC1437 , respectively, into pCM66T_P BAD -RFP (cf. supplementary material 2).

Techniques: Transgenic Assay, Expressing, Polymer

Journal: Journal of Bacteriology

Article Title: The MerR-Like Transcriptional Regulator BrlR Contributes to Pseudomonas aeruginosa Biofilm Tolerance

doi: 10.1128/JB.00765-12

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: Where necessary, Escherichia coli cultures were grown in LB broth in the absence or presence of 1 mM isopropyl-β- d -thiogalactopyranoside (IPTG). table ft1 table-wrap mode="anchored" t5 caption a7 Strain or plasmid Relevant genotype or description Source or reference Escherichia coli DH5α F − φ80d lacZ ΔM15 Δ( lacZYA - argF ) U169 recA1 endA1 hsdR17 (r K − m K + ) phoA supE44 thi - 1 gyrA96 relA1 tonA Invitrogen Corp. BL21 F − ompT hsdS B (r B − m B − ) gal dcm rne131 (DE3) Invitrogen Corp. P. aeruginosa PAO1 PAO1 Wild type B. H. Holloway PAO1/pJN105 PAO1 bearing empty pJN105 vector; Gm r 55 PAO1/pMJT1 PAO1 bearing empty pMJT1 vector; Carb r This study PAO1/pJN- brlR Arabinose-inducible expression of brlR ; Gm r This study PAO1/pMJT- brlR -V5/6×His Arabinose-inducible expression of V5/6×His-tagged brlR ; Carb r This study Δ brlR mutant PAO1 Δ brlR (PA4878) This study Δ brlR /pJN- brlR mutant Arabinose-inducible expression of brlR ; Gm r This study Δ brlR /pMJT1 mutant Δ brlR harboring empty pMJT1, vector control; Carb r This study Δ brlR /pMJT- brlR -N mutant Arabinose-inducible expression of N-terminal DNA binding domain of BrlR (BrlR-N) in Δ brlR ; Carb r This study CF1-2 Isolate from CF patient 53 CF1-8 Isolate from CF patient 53 CF1-8/pJN- brlR Arabinose-inducible expression of brlR in CF1-8; Gm r This study CF1-8 Δ brlR CF1-8 harboring Δ brlR (PA4878), Gm r This study CF1-13 Isolate from CF patient 53 CF1-13 Δ brlR CF1-13 harboring Δ brlR (PA4878); Gm r This study PAO1 attB :: lacZ pminiCTX- lacZ conjugated into PAO1, vector control; Tet r This study PAO1 attB ::PbrlR- lacZ pminiCTX-PbrlR- lacZ conjugated into PAO1; Tet r This study Plasmids pCR2.1-TOPO TA cloning vector; Km r Ap r Invitrogen Corp. pRK2013 Helper plasmid for triparental mating; mob tra Km r 25 pEX18Gm Gene replacement vector; pUC18 MCS oriT sacB Gm r 26 pJN105 Arabinose-inducible gene expression vector; pBRR-1 MCS araC -P BAD Gm r 50 pMJT1 Arabinose-inducible gene expression vector; pUCP18 MCS araC -P BAD Amp/Carb r 35 pET101D Vector for directional cloning and high level V5/6×His fusion protein expression; Amp r Invitrogen Corp. pminiCTX- lacZ attB site-specific integration vector; Tet r 10 pET- brlR -V5/6×His brlR cloned into pET101D This study pJN- brlR brlR cloned into pJN105 This study pMJT- brlR -V5/6×His brlR -V5/6×His cloned into pMJT1 This study pMJT- brlR -N N-terminal DNA binding domain of BrlR cloned into pMJT1 This study pminiCTX-PbrlR- lacZ brlR promoter reporter construct in mini-CTX- lacZ using PbrlR-lacZF/R primers; Tet r This study Open in a separate window Bacterial strains and plasmids used in this study

Techniques: Plasmid Preparation, Expressing, Mutagenesis, Control, Binding Assay, TA Cloning, Gene Expression, Cloning, Clone Assay, Construct

Journal: Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association

Article Title: Pharmaceutical agent cetylpyridinium chloride inhibits immune mast cell function by interfering with calcium mobilization.

doi: 10.1016/j.fct.2023.113980

Figure Lengend Snippet: Fig. 3. CPC effects on mitochondrial Ca2þ in Ag- stimulated RBL mast cells. RBL mast cells were transiently transfected with CEPIA2mt construct, and the mitochondrial Ca2+ levels were measured using a plate reader. Cells were sensitized with IgE for 1 h, pretreated with 0 or 10 μM CPC in BT buffer for 30 min, and exposed to ±0.005 μg/mL Ag with 0 or 10 μM CPC in BT buffer for 1 h. Representative graphs show CEPIA2mt fluorescence immediately following Ag/CPC exposure (A); average background fluores cence, from untransfected cells, was subtracted from all time points prior to plotting. Area under the curve (AUC) was calculated using the background- subtracted fluorescence time-series graph; these values were then normalized to Ag + 0 μM CPC on that day (B). Values presented in (A) are means ± SD for a single experiment with three replicates per treatment. Values presented in (B) are means ± SEM of at least three independent experiments, each experiment was a triplicate. Statistically significant result, as compared to Ag, is represented by ***p < 0.001, determined by an unpaired one-tailed t-test.

Article Snippet: RBL-2H3 cells were transfected with the mitochondrial calcium indicator pCMV CEPIA2mt (a gift from Masamitsu Iino; Addgene plasmid # 58,218; htt p://n2t.net/addgene: 58,218; PRID:Addgene_58,218) (Suzuki et al., 2014) via nucleofection using Amaxa Nucleofector kit T (Lonza), as done in (Weatherly et al., 2018).

Techniques: Transfection, Construct, Fluorescence, One-tailed Test

Journal:

Article Title: The Positive Regulator, TraJ, of the Escherichia coli F Plasmid Is Unstable in a cpxA * Background

doi: 10.1128/JB.184.20.5781-5788.2002

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: Standard genetic techniques were employed ( 46 ). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Genotype or description a Source or reference Bacterial strains MC4100 F − araD139 Δ( argF-lac ) U169 rpsL150 (Str r ) relA1 flb5301 deoC1 ptsF25 rbsR 2 XK1200 Nal r F − lac Δ U124 Δ( nadA araG gal attL ) 34 TR8 MC4100 cpxA :: cam 42 TR20 MC4100 cpxA101 This work TR51 MC4100 cpxR :: spc 42 TR189 MC4100 cpxA101 zii ::Tn 10 λ RS88 [ degP-lacZ ] This work TR981 MC4100 cpxA101 zii ::Tn 10 λ RS88 [ degP-lacZ ] recA This work TR984 MC4100 cpxA101 zii ::Tn 10 λ RS88 [ degP-lacZ ] clpP lonA This work JMR201 MC4100 degP ::Tn 10 A. E. Rizzitello and T. J. Silhavy Plasmids pOX38-Km Km r F tra region, Rep FIA replicon 4 pOX38-Tc Tc r F tra region, Rep FIA replicon 1 pRS27 Tc r 9-kb partial Eco RI F fragmentin pSC101 50 pBAD24 Ap r P BAD cloning vector 16 pBADTraJ traJ coding region fused to P BAD This work pBADTraM traM coding region fused to P BAD This work pBADTraY traY coding region fused to P BAD This work pBR322 General cloning vector New England Biolabs pLD404 Ap r NlpE in pBR322 51 pMC874 Km r lacZ + Δ plac 3 pMCJ211 Km r traJ Ω lacZ FinP + traJ-lacZ reporter plasmid 57 pGEX-KG IPTG-inducible GST fusion expression Amersham Pharmacia Biotech pLJ5-13 Ap r T7 Φ 10-finP fusion in pUC19 19 pSLF20 finP F derivative 24 Open in a separate window a Str r , streptomycin resistant; Nal r , nalidixic acid resistant; Km r , kanamycin resistant; Tc r , tetracycline resistant; Ap r , ampicillin resistant.

Techniques: Plasmid Preparation, Clone Assay, Expressing