Journal: BMC Molecular Biology

Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

doi: 10.1186/1471-2199-12-16

Figure Lengend Snippet: Efficiency of SXT-Bet + SXT-Exo mediated recombination between a PCR-generated DNA fragment and its homologous target on the E. coli chromosome . Panel A: Schematic overview of the chromosomal targeting assay use to score exonuclease + SSAP-mediate recombination efficiency. A dsDNA 'targeting' molecule ( galK<>Cm r ) was synthesized by PCR using two primers sharing (ca. 20 nt) sequence homology to a chloramphenicol resistance cassette at one end; and 50 nt of sequence homology to the 5'-end (ECgalKF1) or 3'-end (ECgalKR1) of the non-essential E. coli galK gene at the other. The purified galK<>Cm r targeting cassette was electroporated into DH10B cells expressing pairs of Exo and SSAP proteins from established arabinose-inducible plasmids. The Exo and SSAP proteins mediate homologous recombination between the galK<>Cm r dsDNA cassette and the galK locus of the E. coli chromosome via 50 bp regions of flanking sequence homology. This creates a mutant E. coli strain that has its galK gene replaced with a chloramphencol resistance cassette. Panel B . Comparison of dsDNA recombination efficiencies of SXT-Bet-Exo with those of RecET and lambda-Bet-Exo. The RecET; lambda-Bet-Exo; SXT-Bet-Exo and SXT-Ssb-Bet-Exo sets of homologous recombination-promoting proteins were expressed from arabinose-inducible (P BAD /araC) plasmids established in E. coli DH10B cells (see Additional File ). pBAD-ETγ: RecE + RecT + lambda-Gam ; pB1E4A: lambda-Bet + lambda Exo; pBex4b1: SXT-Bet + SXT-Exo; pBX2B: SXT-Ssb + SXT-Bet + SXT-Exo; pBAD-28MCS (negative control). Homologous recombination events were scored by plating galK<>Cm r -electroporated cells onto LB-agar with/without chloramphenicol. The dsDNA recombination efficiency was calculated by dividing the number of Cm r colonies by the total number of cells (CFUs) that survived electroporation. 8 replicates were performed; the mean recombination efficiency ± standard deviation is reported.

Article Snippet: The lambda-exo gene was PCR-amplified from bacteriophage lambda cI857 ind 1 Sam7 DNA (NEB) using the exofor1 (TATAA CATATG ACACCGGACATTATCCTG) and exorev3 (TTAT CTCGAG TCGCCATTGCTCCCCAAA) primers, and cloned via NdeI/XhoI into pET32a (Novagen) to create pEE4.

Techniques: Generated, Synthesized, Sequencing, Purification, Expressing, Homologous Recombination, Mutagenesis, Negative Control, Electroporation, Standard Deviation

Journal: BMC Molecular Biology

Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

doi: 10.1186/1471-2199-12-16

Figure Lengend Snippet: Purification of SXT-Exo and lambda-Exo, and determination of their multimericity by size exclusion chromatography . Panel A : Size exclusion chromatogram of purified SXT-Exo protein expressed from plasmid pEA1-1. Panel B : Size exclusion chromatogram of purified lambda-Exo protein expressed from plasmid pEE4. Panel C : 12% polyacrylamide gel (SDS-PAGE) analysis of the SXT-Exo purification procedure and purified SXT-Bet, SXT-Ssb, lambda-Bet and lambda-Exo proteins; lane 1: Benchmark protein ladder (Invitrogen); lane 2: pEA1-1/ E. coli BL21 (DE3) pLysS Rosetta whole cell extract immediately prior to induction; lane 3: whole cell extract 6 hours after induction with IPTG; lane 4: supernatant from cell extract 6 hours post induction; lane 5: purified SXT-Exo; lane 6: purified SXT-Bet expressed from pX28-1; lane 7: purified SXT-Ssb expressed from pSB2; lane 8: purified lambda-Bet expressed from p1DB; lane 9: purified lambda-Exo expressed from pEE4.

Article Snippet: The lambda-exo gene was PCR-amplified from bacteriophage lambda cI857 ind 1 Sam7 DNA (NEB) using the exofor1 (TATAA CATATG ACACCGGACATTATCCTG) and exorev3 (TTAT CTCGAG TCGCCATTGCTCCCCAAA) primers, and cloned via NdeI/XhoI into pET32a (Novagen) to create pEE4.

Techniques: Purification, Size-exclusion Chromatography, Plasmid Preparation, SDS Page

Journal: BMC Molecular Biology

Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

doi: 10.1186/1471-2199-12-16

Figure Lengend Snippet: Qualitative analysis of the metal ion dependence, DNA substrate preferences and mode of digestion of the SXT-Exo alkaline exonuclease . Panel A : Agarose gel showing ability of SXT-Exo to digest linear dsDNA (NdeI-linerized pET28a; lanes 2-5), circularized dsDNA (undigested pET28a; lanes 6 and 7), circularized ssDNA (M13 phage DNA; lanes 8 and 9) in Tris-HCl pH7.4, 50 mM NaCl with/without 10 mM MgCl 2 ; λ-HindIII (NEB) DNA ladder (lane1). Panel B : Agarose gel showing the ability of SXT-Exo and lambda-Exo to digest 5'-phosphorylated linear dsDNA substrates ('unmodified'; lanes 2, 3, 6 and 7), compared with analogous 5'-phosphorylated linear dsDNA substrates containing 3 consecutive phosphorothioate linkages at the 5'-termini of each strand (PT-modified; lanes 4, 5, 8 and 9). The 712 bp 'unmodified' or 'PT-modified' dsDNA substrates (0.1 mg) were incubated at 37°C with lambda-Exo (3 μg) or SXT-Exo (30 μg) in Tris-HCl, (25 mM, pH7.4), 50 mM NaCl, 10 mM MgCl 2 (total volume 40 μl). Aliquots (20 μl) were quenched (20 mM EDTA + 1% SDS) immediately, and after 30 mins, and analyzed on 1% agarose TAE gels. 1 Kb Plus DNA Ladder (Invitrogen; lane 1). Panel C : Agarose gel showing time-course of digestion of 5'-phosphorylated linear dsDNA (NdeI-linearized pET28a, 0.56 pmol) by SXT-Exo (50 pmol of trimers) in Tris-HCl pH7.4, 50 mM NaCl, 10 mM MgCl 2 ; at 37°C, with aliquots removed at times indicated (0-160 minutes; lanes 2-11); 1 Kb Plus DNA Ladder (lane 1).

Article Snippet: The lambda-exo gene was PCR-amplified from bacteriophage lambda cI857 ind 1 Sam7 DNA (NEB) using the exofor1 (TATAA CATATG ACACCGGACATTATCCTG) and exorev3 (TTAT CTCGAG TCGCCATTGCTCCCCAAA) primers, and cloned via NdeI/XhoI into pET32a (Novagen) to create pEE4.

Techniques: Agarose Gel Electrophoresis, Modification, Incubation

Journal: BMC Molecular Biology

Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

doi: 10.1186/1471-2199-12-16

Figure Lengend Snippet: Digestion of fluorescently-labeled annealed oligonucleotide substrates by lambda-Exo . In experiments analogous to those described for SXT-Exo (see Figure 7), the ability of lambda-Exo to digest three different (partially) dsDNA substrates was investigated. In each assay, lambda-Exo (3 pmol of trimers) was incubated at 25°C with 20 pmol of the dsDNA substrate in 50 mM Tris-HCl pH8.0, 5 mM MgCl 2 . Aliquots were removed and quenched at 0, 0.5, 1, 2, 4 and 10 minutes; then analyzed on 7 M urea-TBE denaturing polyacrylamide gels (times indicated above lanes). Gels were scanned for fluorescence, and fluorescence intensities of the bands corresponding to the Cy3-labeled strand were quantified. Panel A : Representative fluorescence-scanned gel image showing time-wise digestion of the 5'-overhang DNA substrate by SXT-Exo. Panel B : Representative gel image showing digestion of the Blunt ended DNA substrate by lambda-Exo. Panel C : Representative gel image showing digestion of the 3'-overhang DNA substrate by lambda-Exo. Panel D : Plot showing the digestion of the three DNA substrates by lambda-Exo over a 10 minute period; reported as the mean percentage ± standard deviation, based on three independent replicates. See materials section for details.

Article Snippet: The lambda-exo gene was PCR-amplified from bacteriophage lambda cI857 ind 1 Sam7 DNA (NEB) using the exofor1 (TATAA CATATG ACACCGGACATTATCCTG) and exorev3 (TTAT CTCGAG TCGCCATTGCTCCCCAAA) primers, and cloned via NdeI/XhoI into pET32a (Novagen) to create pEE4.

Techniques: Labeling, Incubation, Fluorescence, Standard Deviation

Journal: BMC Molecular Biology

Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

doi: 10.1186/1471-2199-12-16

Figure Lengend Snippet: Stimulation of double strand DNA exonuclease activities of SXT-Exo and lambda-Exo by SSAP and Ssb proteins . Panel A . SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) and 2 pmol of the protein indicated in the text (BSA, lambda-Bet, SXT-Bet or SXT-Ssb) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 0.5 mM MnCl 2 ; were incubated at 25°C for 30 mins before EDTA quenching. dsDNA levels were immediately quantified using PicoGreen reagent. The level of DNA digestion by SXT-Exo in the absence of added protein (-) was normalized to a value of 100%. Panel B . In analogous sets of experiments, lambda-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) and 2 pmol of BSA, lambda-Bet, SXT-Bet or SXT-Ssb; in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 5 mM MgCl 2 ; were incubated at 25°C for 10 mins. Digestion levels were normalized to those of lambda-Exo in the absence of added protein (-). See methods section for detailed experimental procedure. Six independent replicates were performed for each experiment, and error bars indicate standard deviation from the mean values. Analysis using ANOVA indicated all results were statistically significant (P < 0.05) when compared to the no-protein control (-), with respective P values indicated above each bar.

Article Snippet: The lambda-exo gene was PCR-amplified from bacteriophage lambda cI857 ind 1 Sam7 DNA (NEB) using the exofor1 (TATAA CATATG ACACCGGACATTATCCTG) and exorev3 (TTAT CTCGAG TCGCCATTGCTCCCCAAA) primers, and cloned via NdeI/XhoI into pET32a (Novagen) to create pEE4.

Techniques: Incubation, Standard Deviation

Journal: PLoS ONE

Article Title: First Description of Natural and Experimental Conjugation between Mycobacteria Mediated by a Linear Plasmid

doi: 10.1371/journal.pone.0029884

Figure Lengend Snippet: M. avium and M. kansasii colonies. (A) PFGE with undigested DNA; (B) Southern blot hybridization with IS 1245 -derived probe. Open arrow indicates pMA100; closed arrow indicates the uncharacterized smaller hybridization band. 88.1 to 88.4 = M . avium ; 88.5 to 88.75 = PCR−IS 1245- negative M . kansasii ; 88.8 to 88.15 = PCR−IS 1245- positive M. kansasii. On the left, Lambda Ladder PFG Marker (NewEngland BioLabs) molecular size markers.

Article Snippet: Besides that, the pMA100 band excised from PFGE agarose gels was treated in separate experiments with 30 U exonuclease III, 30 U exonuclease lambda or 30 U topoiosomerase I (all enzymes from New England BioLabs) for 3 h at 37°C, according to the manufacturer's protocols.

Techniques: Southern Blot, Hybridization, Derivative Assay, Marker

Journal: PLoS ONE

Article Title: First Description of Natural and Experimental Conjugation between Mycobacteria Mediated by a Linear Plasmid

doi: 10.1371/journal.pone.0029884

Figure Lengend Snippet: (A) PFGE with undigested DNAs from M. avium 88.3 (1) and M. kansasii 88.8 (2) under different switch times, indicated below each figure; (B) pMA100 extracted from PFGE gels and treated with exonuclease III (3) or exonuclease lambda (4); (C) pMA100 extracted from PFGE gels and treated (+) or not (-) with topoisomerase I; (D) DNA prepared with (+) or without (-) adding proteinase K to the lysis buffer; (E) same as in (D) in PFGE gels and running buffer prepared with 0.2% SDS. λ: DNA concatemers of the bacteriophage λ genome.

Article Snippet: Besides that, the pMA100 band excised from PFGE agarose gels was treated in separate experiments with 30 U exonuclease III, 30 U exonuclease lambda or 30 U topoiosomerase I (all enzymes from New England BioLabs) for 3 h at 37°C, according to the manufacturer's protocols.

Techniques: Lysis