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    New England Biolabs nla iv sites
    <t>Nla</t> IV restriction affects plasmid transformation in N. meningitidis MC58 wildtype and not the Nla IV null mutant. The Y axis shows the number of resistant (erythromycin) CFU/total 10 10 CFU on a log scale. Along the X-axis are the different DNA substrates (10 ng/ml) with altered numbers of Nla IV restriction sites shown. pDV4-a harbours two and three Nla IV restriction sites more than pDV4-b and pDV4-c, respectively. For further details of the restriction profiles and <t>DUS</t> locations in the transforming DNA plasmids please consult Figure 1 and Table 2 . Statistically significant differences in transformation frequencies between the Nla IV null mutant and wildtype backgrounds are indicated above the columns, ***equals p≤0.001 in a two tailed paired student’s t-test.
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    Nla IV restriction affects plasmid transformation in N. meningitidis MC58 wildtype and not the Nla IV null mutant. The Y axis shows the number of resistant (erythromycin) CFU/total 10 10 CFU on a log scale. Along the X-axis are the different DNA substrates (10 ng/ml) with altered numbers of Nla IV restriction sites shown. pDV4-a harbours two and three Nla IV restriction sites more than pDV4-b and pDV4-c, respectively. For further details of the restriction profiles and DUS locations in the transforming DNA plasmids please consult Figure 1 and Table 2 . Statistically significant differences in transformation frequencies between the Nla IV null mutant and wildtype backgrounds are indicated above the columns, ***equals p≤0.001 in a two tailed paired student’s t-test.

    Journal: PLoS ONE

    Article Title: Restriction and Sequence Alterations Affect DNA Uptake Sequence-Dependent Transformation in Neisseria meningitidis

    doi: 10.1371/journal.pone.0039742

    Figure Lengend Snippet: Nla IV restriction affects plasmid transformation in N. meningitidis MC58 wildtype and not the Nla IV null mutant. The Y axis shows the number of resistant (erythromycin) CFU/total 10 10 CFU on a log scale. Along the X-axis are the different DNA substrates (10 ng/ml) with altered numbers of Nla IV restriction sites shown. pDV4-a harbours two and three Nla IV restriction sites more than pDV4-b and pDV4-c, respectively. For further details of the restriction profiles and DUS locations in the transforming DNA plasmids please consult Figure 1 and Table 2 . Statistically significant differences in transformation frequencies between the Nla IV null mutant and wildtype backgrounds are indicated above the columns, ***equals p≤0.001 in a two tailed paired student’s t-test.

    Article Snippet: The hybrid plasmids pOHA-0-DUS and pOHA-Single from a previous study containing the naturally DUS-less neisserial pilus biogenesis gene pilG harbouring a selective marker encoding erythromycin (erm) resistance , were used to make all constructs employed, varying in DUS, Nla IV sites and homologous region as shown in . pOHA-0-DUS and pSingle were trimmed with Bam HI (recognition sequence GGATCC) (New England Biolabs) and recircularised with T4 Ligase (New England Biolabs) or polished with Phusion polymerase (New England Biolabs) following Bam HI restriction and recircularised to generate the plasmids -b, -c and -d versions of pOHA-0-DUS and pOHA13, respectively.

    Techniques: Plasmid Preparation, Transformation Assay, Mutagenesis, Two Tailed Test

    Sequence characteristics of transforming DNA. A. Variable DUS positions and restriction profiles in transforming DNA plasmids. Transforming DNA plasmids are based on pilG (black) interrupted by an erythromycin resistance insert, ermC , (gray). DUS ( 5′- ATGCCGTCTGAA-3′ ) or reverse complimentary DUS ( 5′-TTCAGACGGCAT-3′ ) are inserted in three different positions A, B and C as marked above the bar. All numbers refer to the nucleotide position following the start codon (1) of pilG. The 137 nucleotide long Bam HI-fragment which is removed in pDV-b, pDV-c versions is shown in white with black stripes. B. Nla IV restriction profile of rpoB and location of selective SNP. The homologous 723 nt long PCR fragment of an internal part of the meningococcal rpoB gene used in transformation contains two Nla IV restriction sites on both sides of the selective SNP responsible for rifampicin resistance in the recipient.

    Journal: PLoS ONE

    Article Title: Restriction and Sequence Alterations Affect DNA Uptake Sequence-Dependent Transformation in Neisseria meningitidis

    doi: 10.1371/journal.pone.0039742

    Figure Lengend Snippet: Sequence characteristics of transforming DNA. A. Variable DUS positions and restriction profiles in transforming DNA plasmids. Transforming DNA plasmids are based on pilG (black) interrupted by an erythromycin resistance insert, ermC , (gray). DUS ( 5′- ATGCCGTCTGAA-3′ ) or reverse complimentary DUS ( 5′-TTCAGACGGCAT-3′ ) are inserted in three different positions A, B and C as marked above the bar. All numbers refer to the nucleotide position following the start codon (1) of pilG. The 137 nucleotide long Bam HI-fragment which is removed in pDV-b, pDV-c versions is shown in white with black stripes. B. Nla IV restriction profile of rpoB and location of selective SNP. The homologous 723 nt long PCR fragment of an internal part of the meningococcal rpoB gene used in transformation contains two Nla IV restriction sites on both sides of the selective SNP responsible for rifampicin resistance in the recipient.

    Article Snippet: The hybrid plasmids pOHA-0-DUS and pOHA-Single from a previous study containing the naturally DUS-less neisserial pilus biogenesis gene pilG harbouring a selective marker encoding erythromycin (erm) resistance , were used to make all constructs employed, varying in DUS, Nla IV sites and homologous region as shown in . pOHA-0-DUS and pSingle were trimmed with Bam HI (recognition sequence GGATCC) (New England Biolabs) and recircularised with T4 Ligase (New England Biolabs) or polished with Phusion polymerase (New England Biolabs) following Bam HI restriction and recircularised to generate the plasmids -b, -c and -d versions of pOHA-0-DUS and pOHA13, respectively.

    Techniques: Sequencing, Polymerase Chain Reaction, Transformation Assay

    The influence of DUS location on transformation. The Y axis shows the number of erythromycin-resistant CFU/total CFU 10 8 on a log scale. Along the X-axis are shown the transforming DNA (1 µg/ml) that differ in Nla IV restriction profiles and DUS location. For further details on the DNA plasmid templates please consult Figure 1 and Table 2 . Statistically significant values are indicated above the columns, **equals p≤0.05 and ***equals p≤0.001 in a two-tailed paired student’s t tests.

    Journal: PLoS ONE

    Article Title: Restriction and Sequence Alterations Affect DNA Uptake Sequence-Dependent Transformation in Neisseria meningitidis

    doi: 10.1371/journal.pone.0039742

    Figure Lengend Snippet: The influence of DUS location on transformation. The Y axis shows the number of erythromycin-resistant CFU/total CFU 10 8 on a log scale. Along the X-axis are shown the transforming DNA (1 µg/ml) that differ in Nla IV restriction profiles and DUS location. For further details on the DNA plasmid templates please consult Figure 1 and Table 2 . Statistically significant values are indicated above the columns, **equals p≤0.05 and ***equals p≤0.001 in a two-tailed paired student’s t tests.

    Article Snippet: The hybrid plasmids pOHA-0-DUS and pOHA-Single from a previous study containing the naturally DUS-less neisserial pilus biogenesis gene pilG harbouring a selective marker encoding erythromycin (erm) resistance , were used to make all constructs employed, varying in DUS, Nla IV sites and homologous region as shown in . pOHA-0-DUS and pSingle were trimmed with Bam HI (recognition sequence GGATCC) (New England Biolabs) and recircularised with T4 Ligase (New England Biolabs) or polished with Phusion polymerase (New England Biolabs) following Bam HI restriction and recircularised to generate the plasmids -b, -c and -d versions of pOHA-0-DUS and pOHA13, respectively.

    Techniques: Transformation Assay, Plasmid Preparation, Two Tailed Test