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    How To Transfer A Mobilizable Plasmid From A Cell Without A Conjugative Plasmid?

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    m86544422
    Here are some potential reasons why no colonies were obtained after using transformation or conjugation: - The bacterial cells may not have been competent for DNA uptake during transformation. Not all cells are naturally competent. - The DNA used for transformation was not in a form that can enter the cells (e.g. linear DNA instead of supercoiled plasmid). - Inhibitors may have been present in the DNA preparation or solution that prevented DNA entry into cells. - The selection antibiotic was not present or not at a high enough concentration to select for transformants/transconjugants. - The plasmid DNA or donor cells were no longer viable or active at the time of the procedure due to improper storage or handling.

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    How To Transfer A Mobilizable Plasmid From A Cell Without A Conjugative Plasmid?

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    m86544422
    42 views14 pages
    Here are some potential reasons why no colonies were obtained after using transformation or conjugation: - The bacterial cells may not have been competent for DNA uptake during transformation. Not all cells are naturally competent. - The DNA used for transformation was not in a form that can enter the cells (e.g. linear DNA instead of supercoiled plasmid). - Inhibitors may have been present in the DNA preparation or solution that prevented DNA entry into cells. - The selection antibiotic was not present or not at a high enough concentration to select for transformants/transconjugants. - The plasmid DNA or donor cells were no longer viable or active at the time of the procedure due to improper storage or handling.

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    Apuntes ingenyeria genètica uab

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    09.03.23

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    Here are some potential reasons why no colonies were obtained after using transformation or conjugation: - The bacterial cells may not have been competent for DNA uptake during transformation. Not all cells are naturally competent. - The DNA used for transformation was not in a form that can enter the cells (e.g. linear DNA instead of supercoiled plasmid). - Inhibitors may have been present in the DNA preparation or solution that prevented DNA entry into cells. - The selection antibiotic was not present or not at a high enough concentration to select for transformants/transconjugants. - The plasmid DNA or donor cells were no longer viable or active at the time of the procedure due to improper storage or handling.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    42 views14 pages

    How To Transfer A Mobilizable Plasmid From A Cell Without A Conjugative Plasmid?

    Uploaded by

    m86544422
    Here are some potential reasons why no colonies were obtained after using transformation or conjugation: - The bacterial cells may not have been competent for DNA uptake during transformation. Not all cells are naturally competent. - The DNA used for transformation was not in a form that can enter the cells (e.g. linear DNA instead of supercoiled plasmid). - Inhibitors may have been present in the DNA preparation or solution that prevented DNA entry into cells. - The selection antibiotic was not present or not at a high enough concentration to select for transformants/transconjugants. - The plasmid DNA or donor cells were no longer viable or active at the time of the procedure due to improper storage or handling.

    Copyright:

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    of 14

    HOW TO TRANSFER A MOBILIZABLE

    PLASMID FROM A CELL WITHOUT A


    CONJUGATIVE PLASMID?
    TRIPARENTAL MATING

    “Molecular Genetics of Bacteria” L. Snyder and W, Champness. 2003. Third edition


    WHICH STRAINS ARE ABLE TO
    MOBILIZE PLASMIDS?
    • Carriers of plasmids Mob+ Tra+
    • Carriers of plasmids Mob- Tra+
    • Strains carrying tra genes:
    RP4 (incPa) integrated in its bacterial chromosome
    Hfr cells: High frequency of recombination

    Chromosome fragment
    integrated into F factor

    Detachment of F
    factor carrying a
    chromosome
    fragment

    LEDERBERG AND TATUM


    EXPERIMENT (1946)
    PHEROMONE-INDUCIBLE CONJUGATION IN
    GRAM-POSITIVE BACTERIA

    • Present in some Gram-positive conjugation systems.

    • One of the most studied: Enterococcus faecalis

    Pheromones: hydrophobic peptides obtained from the


    processing of the secreted lipoprotein signal peptide

    Some plasmids exhibit pheromone detection systems


    LIPOPROTEINS SYNTHESIZED BY
    THE CHROMOSOME OF E. faecalis
    pPD1 plasmid pheromone

    pAD1 plasmid pheromone

    pCF10 plasmid pheromone

    The cut takes place by


    endoproteases on the
    membrane and a transport
    system is needed for
    Recipient cell exporting the oligopeptide (7-
    8 aa C-terminus) to the
    outside.
    “Molecular Genetics of Bacteria” L. Snyder and W, Champness. 2003. Third edition
    CONJUGATIVE PLASMIDS WITH
    PHEROMONE SENSORS
    Pheromone sensor system

    Transcriptonal repressor of
    conjugation-encoding
    genes

    Donor cell

    “Molecular Genetics of Bacteria” L. Snyder and W, Champness. 2003. Third edition


    DETECTION OF RECIPIENT CELLS

    Oligopeptide permease

    Pheromone cAD1 inhibits TraA repressor:


    conjugation genes are expressed

    Aggregation substance

    Donor cell
    Negative induction control,
    TraA is the regulator and
    cAD1 is the inductor
    “Molecular Genetics of Bacteria” L. Snyder and W, Champness. 2003. Third edition
    CONJUGATION IN GRAM-
    POSITIVE BACTERIA

    Relaxosome
    formation, ssDNA,
    transference and
    replication

    Donor cell

    “Molecular Genetics of Bacteria” L. Snyder and W, Champness. 2003. Third edition


    Model of T4SS proteins in
    Gram-positive bacteria
    (PMID: 24129002)
    EXCLUSION SYSTEM
    Peptide iAD1 inhibits receptor TraC to avoid the
    transferce of the newly incorporated plasmid until a new
    receptor cell increases the cAD1 pheromone
    concentration

    Pheromone shutdown: TraB


    avoids cAD1 exportation; the
    cell does not need the
    incorporation of the same
    plasmid again

    Other pheromones may be


    produced to incorporate
    Recipient cell other plasmids

    “Molecular Genetics of Bacteria” L. Snyder and W, Champness. 2003. Third edition


    We have a multicopy plasmid and we want
    to include it within a Salmonella enterica
    strain.

    How can you do it?

    What do you need to consider?


    Things to be considered…

    IF TRANSFORMATION IS USED...

    Any DNA could enter into the cells?


    Do all bacteria need the same transformation requirements?

    Do vectors enter better than lineal DNA?


    Do all vectors enter with the same efficiency?
    Things to be considered…
    IF CONJUGATION IS USED...

    Who is the donor strain and who the recipient in a conjugation?

    Is it a conjugative, a mobilizable, or a non-mobilizable vector?

    Do we need a helper strain or helper vector?


    Is recombination necessary to stabilize the included plasmid?
    We use a transformation or a
    conjugation procedure, but we obtain
    no colonies.
    What has it happened?

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