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    Six Cu resistant bacterial strains namely Cu1,2,3,4,5 and 6 were isolated from tanneries effluents in Kasoor industrial area .Their Minimum Inhibitory Concentration(MIC) was determined by growing them on LB medium supplemented with Cu++ in CuSo4 form, with gradual increase in Cu++ concentration. The MIC of Cu1, 2, 3, 4, 5 and 6 was 300,500,600,500,800 and 500 ppm respectively. These strains were identified though 16S rRNA ribotyping by using universal primers for 16S rRNA partial gene. The amplified DNA product was ligated with plasmid pTZ57R/T, transformed in DH5α host cells and sequenced. The strains Cu1, 2, 3 and 4 were Bacilli,Cu5 staphylococcus and Cu6 Enterococcus.

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    Six Cu resistant bacterial strains namely Cu1,2,3,4,5 and 6 were isolated from tanneries effluents in Kasoor industrial area .Their Minimum Inhibitory Concentration(MIC) was determined by growing them on LB medium supplemented with Cu++ in CuSo4 form, with gradual increase in Cu++ concentration. The MIC of Cu1, 2, 3, 4, 5 and 6 was 300,500,600,500,800 and 500 ppm respectively. These strains were identified though 16S rRNA ribotyping by using universal primers for 16S rRNA partial gene. The amplified DNA product was ligated with plasmid pTZ57R/T, transformed in DH5α host cells and sequenced. The strains Cu1, 2, 3 and 4 were Bacilli,Cu5 staphylococcus and Cu6 Enterococcus.

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    25 views16 pages

    Determination of Copper Resistance in Bacteria

    Uploaded by

    Soumble
    Six Cu resistant bacterial strains namely Cu1,2,3,4,5 and 6 were isolated from tanneries effluents in Kasoor industrial area .Their Minimum Inhibitory Concentration(MIC) was determined by growing them on LB medium supplemented with Cu++ in CuSo4 form, with gradual increase in Cu++ concentration. The MIC of Cu1, 2, 3, 4, 5 and 6 was 300,500,600,500,800 and 500 ppm respectively. These strains were identified though 16S rRNA ribotyping by using universal primers for 16S rRNA partial gene. The amplified DNA product was ligated with plasmid pTZ57R/T, transformed in DH5α host cells and sequenced. The strains Cu1, 2, 3 and 4 were Bacilli,Cu5 staphylococcus and Cu6 Enterococcus.

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    of 16

    DETERMINATION OF

    COPPER RESISTANCE
    IN BACTERIA
    Soumble Zulfiqar
    and
    A. R. Shakoori
    School of Biological Sciences,
    University of the Punjab Lahore.

    Determination Of Copper Resistance In


    Bacteria

    COPPER
    Cu
    Cu

    enzymes bioremediation

    Highly toxic (If in excess amount )

    Cu

    Cu

    Cu

    Binds to proteins and nucleic acids and can


    cause the oxidation of lipids and proteins

    Cu

    Cu

    Essential cofactor for more than 30

    Cu

    Micronutrient

    Cu

    Determination Of Copper Resistance In

    METHODOLOGY
    Isolation of copper resistant bacterial
    strains
    from effluents of Kasoor
    industrial area
    Designated as Cu1,Cu2, Cu3, Cu4, Cu5
    & Cu6
    Identification of the bacterial strains
    through ribotyping
    Methodology cont.

    Determination Of Copper Resistance In


    Bacteria

    RIBOTYPING
    Variation in chromosome as a whole likely
    to be reflected in variation in specific
    genes
    It uses so-called universal probes
    targeted at specific conserved domains of
    ribosomal RNA coding sequences,
    allowing characterization with only limited
    sequence information.
    Methodology cont.

    Determination Of Copper Resistance In


    Bacteria

    RIBOSOMAL GENES
    Several different copies at different loci within
    the genome
    Highly conserved in microbes.
    The genetic information coding for rRNA vary
    much less within bacteria of the same strain
    than between bacterial strains.
    This characteristic allows for a greater ability to
    distinguish between different bacterial strains.
    Methodology cont.

    Determination Of Copper Resistance In


    Bacteria

    Genomic DNA Isolation


    Polymerase Chain Reaction (PCR) of 16srRNA partial
    gene(500bp)
    Primers:
    RS 1
    RS 3

    5 AAACTC/TAAAG/TGAATTGACGG
    5 ACGGGCGGTGTGTA/GC

    PCR Reaction:
    30 Cycles
    94C

    94C

    3min

    30sec

    55C

    72C

    72C

    1min

    4-5min

    30sec
    Methodology cont.

    Determination Of Copper Resistance In


    Bacteria

    Ligation in pTZ57R/T
    Transformation of the construct in DH5
    Confirmation of the transformation through
    Digestion Analysis
    Sequencing of the insert-partial 16srRNA gene
    Nucleotide nucleotide BLAST analysis
    Determination
    of
    Minimum
    Inhibitory
    Concentration (MIC)

    Determination Of Copper Resistance In


    Bacteria

    GENOMIC DNA ISOLATION


    M

    Cu Cu Cu Cu Cu Cu
    1 2 3 4 5 6

    23 kb

    Results cont.

    Determination Of Copper Resistance In


    Bacteria

    POLYMERASE CHAIN REACTION

    (16srRNA partial gene-500bp)


    Cu Cu Cu Cu Cu Cu
    1

    5 6

    500bp
    Results cont.

    Determination Of Copper Resistance In


    Bacteria

    SMALL SCALE PLASMID ISOLATION


    M

    Cu Cu Cu Cu Cu Cu
    1 2 3 4 5 6

    3.386kb

    Determination Of Copper Resistance In


    Bacteria

    DOUBLE DIGESTION
    Lane 1,2,3 & 4 cut with Xba &BamH
    Lane 5&6 cut with EcoR & Hind

    Cu Cu Cu Cu Cu Cu M
    1 2 3
    4 5
    6

    Results cont.

    Determination Of Copper Resistance In


    Bacteria

    STRAINS IDENTIFIED
    Cu 1

    Bacillus

    Cu 2

    Bacillus

    Cu 3

    Bacillus pumilus

    Cu 4

    Bacillus

    Cu 5

    Staphylococcus

    Cu 6

    Enterococcus faecium
    Results cont.

    Determination Of Copper Resistance In


    Bacteria

    MINIMUM INHIBITORY CONCENTRATION


    Strain

    MIC(ppm)

    Cu 1

    Bacillus sp.

    300

    Cu 2

    Bacillus sp.

    500

    Cu 3

    Bacillus pumilus

    600

    Cu 4

    Bacillus sp.

    500

    Cu 5

    Staphylococcus
    sp.

    800

    Cu 6

    Enterococcus
    faecium

    500

    Determination Of Copper Resistance In


    Bacteria

    FUTURE PLANS
    Determination
    of
    copper
    uptakeabsorption and adsorption-in the copper
    resistant strains
    Determination
    of
    Copper
    influx
    mechanism in prokaryotes

    QUESTIONS
    AND
    ANSWERS

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