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    Materi 5

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    Rafif Abdikah Utama
    Plasmids are small, circular DNA molecules that can replicate independently of the host chromosome. They are commonly used in molecular biology to clone and amplify DNA sequences. The document discusses plasmid isolation, transformation of bacterial cells, and shuttle vectors that allow plasmids to replicate in both bacteria and yeast. Key steps in plasmid isolation include cell lysis, denaturation and renaturation of DNA, and separation of plasmids from bacterial chromosomes based on their unique physical properties like supercoiling. Transformation involves making bacterial cells competent to take up foreign DNA by creating pores in the cell membrane, then introducing the plasmid and giving a heat shock to allow DNA entry.

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    Materi 5

    Uploaded by

    Rafif Abdikah Utama
    16 views29 pages
    Plasmids are small, circular DNA molecules that can replicate independently of the host chromosome. They are commonly used in molecular biology to clone and amplify DNA sequences. The document discusses plasmid isolation, transformation of bacterial cells, and shuttle vectors that allow plasmids to replicate in both bacteria and yeast. Key steps in plasmid isolation include cell lysis, denaturation and renaturation of DNA, and separation of plasmids from bacterial chromosomes based on their unique physical properties like supercoiling. Transformation involves making bacterial cells competent to take up foreign DNA by creating pores in the cell membrane, then introducing the plasmid and giving a heat shock to allow DNA entry.

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    Plasmids are small, circular DNA molecules that can replicate independently of the host chromosome. They are commonly used in molecular biology to clone and amplify DNA sequences. The document discusses plasmid isolation, transformation of bacterial cells, and shuttle vectors that allow plasmids to replicate in both bacteria and yeast. Key steps in plasmid isolation include cell lysis, denaturation and renaturation of DNA, and separation of plasmids from bacterial chromosomes based on their unique physical properties like supercoiling. Transformation involves making bacterial cells competent to take up foreign DNA by creating pores in the cell membrane, then introducing the plasmid and giving a heat shock to allow DNA entry.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    16 views29 pages

    Materi 5

    Uploaded by

    Rafif Abdikah Utama
    Plasmids are small, circular DNA molecules that can replicate independently of the host chromosome. They are commonly used in molecular biology to clone and amplify DNA sequences. The document discusses plasmid isolation, transformation of bacterial cells, and shuttle vectors that allow plasmids to replicate in both bacteria and yeast. Key steps in plasmid isolation include cell lysis, denaturation and renaturation of DNA, and separation of plasmids from bacterial chromosomes based on their unique physical properties like supercoiling. Transformation involves making bacterial cells competent to take up foreign DNA by creating pores in the cell membrane, then introducing the plasmid and giving a heat shock to allow DNA entry.

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    of 29

    Isolasi Plasmid,

    transformasi,
    dan sekuensing

    Umi Baroroh, S.Si.,


    M.Biotek.
    Plasmids

    Small circular DNA molecules that replicate independently of the


    host chromosomes

    Indispensable tools that allow molecular biologists to obtain


    essentially unlimited amounts of a DNA sequence
    Plasmid
    Circular and double-stranded

    Plasmid size varies from 1 to over 100 kbp

    The number of identical plasmids within a single


    cell can be zero, one, or even hundreds under some
    circumstances.
    • Fertility-F-plasmids, facilitate bacterial
    conjugation

    • Resistance-(R)plasmids, which
    Plasmid contain genes that can build a
    resistance against antibiotics or poisons
    Classification • Col-plasmids, which contain genes that
    based on its code for bacteriocins, protein that can kill
    other bacteria
    function • Degradative plasmids, which enable the
    digestion of unusual substances, e.g.,
    toluene or salicylic acid
    • Virulence plasmids, which turn the
    bacterium into a pathogen.
    Plasmids used in molecular biology have been constructed in the lab

    Molecular cloning

    Enzymes are used to insert desired


    pieces of foreign DNA into plasmids

    Bacterial cells are transformed with


    the plasmids. Copies of the
    plasmids are purified from bacteria.
    https://www.youtube.com/watch?v=SdqJFA6mOkI
    We are using plasmids that have been termed shuttle vectors,
    because they can be propagated in either bacteria or yeast

    non-pathogenic strain Saccharomyces cerevisiae


    of Escherichia coli deletion strains

    Plasmids are propagated in Plasmid-encoded genes


    bacteria, which grow quickly are expressed in yeast,
    and maintain multiple copies and phenotypes are
    of the plasmids analyzed

    Shuttle vectors have origins of replication and selectable markers


    for propagation in both bacteria and yeast
    The pBG1805*-derived plasmids are complex vectors designed
    to express S. cerevisiae ORFs

    5 6 1 URA3

    2 β-lactamase gene
    3
    ORF goes 3 pBR322 ori
    here 1
    2 4 yeast 2 µm origin

    5 GAL1 promoter
    4
    6 C-terminal tags

    *Plasmid names begin with a lower case “p”


    pYES2.1-based plasmids used for S. pombe ORFs have many
    elements in common with pBG1805-based plasmids

    1 URA3 3 pBR322 ori 5 GAL1 promoter

    2 β-lactamase gene 4 yeast 2 µm origin 6 7 C-terminal tags

    5 6 5
    7

    3
    4
    ORF goes
    ORF goes 3
    here 1
    here
    2

    2
    1
    4

    pBG1805 (6573 bp) pYES2.1 (5886 bp)


    used for S. cerevisiae ORFs used for S. pombe ORFs
    Plasmid isolation is based on their distinctive physical
    properties

    Plasmids are much smaller than bacterial


    chromosomes

    Plasmids are supercoiled in their native form

    Supercoiling allows plasmids to renature quickly


    after they are denatured

    Plasmids used in molecular biology are highly engineered and


    contain elements of use to researchers
    • Miniprep
    • Can be used to quickly find out whether the
    Methods plasmid is correct in any of several bacterial
    clones. The yield is a small amount of
    to isolate impure plasmid DNA, which is sufficient for
    analysis by restriction digest and for some
    plasmid cloning techniques.
    • Maxiprep/bulkprep
    DNA • Much larger volumes of bacterial
    suspension are grown from which a maxi-
    prep can be performed. Essentially this is a
    scaled-up miniprep followed by additional
    purification. This results in relatively large
    amounts (several micrograms) of very pure
    plasmid DNA.
    Plasmid isolation from bacteria relies on their unique physical
    properties

    MANY different,
    well-folded proteins

    contains 1-2 copies of large


    (>Mbp) , circular
    bacterial DNA
    Bacterial cell with
    complexed with
    plasmids
    proteins

    Multiple copies of small


    (5-15 kbp) plasmids

    Isolation involves sequential denaturation and renaturation steps


    Cells are first treated with base and a detergent

    Proteins denature
    irreversibly

    Chromosomal DNA
    denatures—will have
    difficulty renaturing
    because of its length
    breaks open membrane and many proteins
    and denatures both DNA complexed to it
    and proteins

    Plasmids denature, but


    strands stay together
    because of supercoiling
    Extract is neutralized to allow DNA molecules to renature

    Plasmids
    renature and are
    suspended in the
    SUPERNATANT
    following
    centrifugation

    Proteins and
    chromosomal DNA form
    aggregate irreversibly,
    forming a PRECIPITATE
    that can be collected by
    centrifugation

    When isolating plasmids, use a micropipette to remove the


    supernatant for further processing steps
    Plasmid DNA isolation
    1. Inactivation of bacteria
    2. Lysis of cells/ denaturation of DNA
    3. Precipitation of DNA
    4. Separate plasmid DNA from
    contaminants
    5. Precipitation of Plasmid DNA
    6. Precipitation of proteins
    7. Precipitate Plasmid DNA
    Bacterial Transformation
    • Transformation is the genetic alteration of a cell resulting from the
    uptake and expression of foreign genetic material (DNA). i.e. the act
    of putting foreign DNA into a bacterial cell

    • Occurs in nature, but rarely

    • If the foreign DNA has an origin of replication recognized by the host


    cell DNA polymerases, the bacteria will replicate the foreign DNA
    along with their own DNA.The origin of replication (also called the
    replication origin) is a particular

    sequence in a genome at which

    replication is initiated
    https://www.youtube.com/watch?v=w_fZmiIAdco
    Bacterial Competence
    • Not all bacteria are capable of taking up DNA from the
    surrounding environment. Such bacteria are made artificially
    competent. This is achieved by using chemicals and electrical
    pulses.
    • Chemicals- The cells are chilled and made permeable in the
    presence of calcium phosphate. They are then incubated with
    the DNA and provided with a heat shock treatment that causes
    the DNA to enter the cells.
    • Electroporation- The bacterial cells are subjected to electrical
    pulses to make them permeable and cause the DNA to enter
    into cells.
    Atrificial competency
    • SinceDNA is a very hydrophilic molecule, it won't normally
    pass through a bacterial cell's membrane.
    • Inorder to make bacteria take in the plasmid, they must
    first be made "competent" to take up DNA.
    • Thisis done by creating small holes in the bacterial cells by
    different methods.
    • Cells
    that are undergoing very rapid growth are made
    competent more easily than cells in other stages of growth.
    1- Competency
    • Label two sterile microtubes: one “+”and the other “-”.
    • Using a disposable pipette, add 250 μl of 50mM CaCl2 solution
    to each tube (“+” and “-”) and place them both on ice.
    • Add 10 µl of ampicillin resistant plasmid
    directly into the CaCl2 in “+” tube.
    • Return the “+” tube to the ice.
    DO NOT add the plasmid to your "-" tube. Incubate both tubes
    on ice for 15 minutes.

    Ca2+ neutralizes the negative charges


    on the phosphate backbone of the DNA,
    so that the DNA is not repulsed from the
    phospholipids of the cell membrane.
    This allows the DNA to more easily pass
    1- Transformation
    TRANSFORMATION EFFICIENCY
    • After incubation count the colonies on each plate.
    • Calculate the transformation efficiency
    • Transformation Efficiency = no. of colonies observed/ µg
    Plasmid
    THANK YOU

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