Bacterial Transformation - Paulding County School District ... Calculating Transformation Efficiency...

Click here to load reader

  • date post

    04-Jun-2020
  • Category

    Documents

  • view

    1
  • download

    0

Embed Size (px)

Transcript of Bacterial Transformation - Paulding County School District ... Calculating Transformation Efficiency...

  • Bacterial

    Transformation and Plasmid Purification

    Chapter 5: Background

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 2

    History of Transformation and Plasmids

     Bacterial methods of DNA transfer

    – Transformation: when bacteria take up DNA from their

    environment

    – Conjugation: process of transferring DNA by a pilus

    (bridge) from one bacteria to another

    – Transduction: when bacterial DNA is transferred from

    one bacteria to another by viruses

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 3

    Origin of Plasmids

     Joshua Lederberg and William Hayes

    independently discovered plasmids

    while studying conjugation – 1952 Lederberg proposed the name plasmid

    – 1961 Tsutomu Watanabe and Toshio

    Fukasawa found that some plasmids carried

    antibiotic resistance genes

    – 1962 Allan Campbell determined that plasmids

    were circular

    – 1973 Peter Lobban proposed using restriction

    enzymes to help recombine DNA

    – 1973 Stanley Cohen, Annie Chang, Herbert

    Boyer, and Robert Helling published a paper

    describing how to construct a functional

    plasmid

    – 1976 Herbert Boyer and Robert Swanson

    founded Genentech using plasmids to

    manufacture insulin

    – 2009 Genentech was sold to Roche for $46

    billion

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 4

    Plasmids: Structure and Function

     Most are extrachromosomal loops of

    DNA that can self-replicate in the

    cytosol of bacteria

    – They have an origin of replication (ori

    on the map)

    – Are designated with a “p” in the name

    – Have genes that code for proteins.

    They are symbolized by an arrow in the

    direction of transcription

    • Genes are preceded by a promoter

    – The location for RNA polymerase to

    bind

    • They are followed by a terminator

    – The location that causes the

    polymerase to stop transcribing

    – Number of plasmids ranges from 5 to

    1,000 per bacterial cell

    • Low copy number plasmids

    • High copy number plasmids

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 5

    Plasmid Uses

     Two main uses

    – To express recombinant

    proteins

    – To house genes that have

    been cloned

    • These can then be

    placed into other

    organisms (e.g. corn)

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 6

    Modern Plasmids

     Plasmids are constructed to make cloning easy.

    They have an area called a multiple cloning site

    (MCS) that has a series of unique restriction enzyme

    recognition sites

    – This MCS is used to open up the plasmid to receive the

    gene of interest

    Plasmid with a gene

    (red) inserted into

    the MCS (green)

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 7

    Recombinant DNA Using Plasmids

     Steps

    – Extract and purify plasmid

    and DNA of interest

    – Digest plasmid and DNA

    of interest with restriction

    enzymes

    • PCR can be used to

    amplify gene of interest

    – Mix the two different DNA

    fragments together and

    add DNA ligase

    – Transform plasmid into

    host cell

    – Grow and select for cells

    that have insert

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 8

    Transcriptional Regulation of Plasmids

     How operons work

    – Jacob and Monod in 1961

    discovered how the lac

    operon work in bacteria

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 9

    Transcriptional Regulation of Plasmids

     How pBAD operon works

    – An operon in which

    arabinose is the inducer

    instead of lactose

    • Different operons have

    different inducers

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 10

    Transcriptional Regulation of Plasmids

     If the three genes BAD are

    cut out by restriction

    enzymes and GFP is

    ligated in their place, a

    recombinant operon is

    produced that expresses

    GFP

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 11

    Other Types of Plasmids

     Shuttle plasmids

    – Plasmids that can be inserted into bacteria initially to be

    cloned, then transformed into eukaryotic cells once

    duplicated and isolated

    • For example, to grow in E. coli, a plasmid needs a prokaryotic

    origin of replication and an antibiotic-resistant gene

    • To grow in a eukaryote, it would need a eukaryotic origin of

    replication, a sequence for a poly A tail, a promoter, and a

    terminator sequence that would function in a eukaryotic cell

     Ti plasmid

    – Found naturally in Agrobacterium tumefaciens

    – Causes crown gall disease in plants

    – Can be modified to carry genes of interest into plants

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 12

    Transforming Cells

     Two major methods of transformation

    – Calcium chloride

    – Electroporation

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 13

    Calcium Chloride Transformation Steps

     Suspend bacterial colonies in 50 mM (0.05 M)

    calcium chloride

     Add plasmid DNA

     Place tubes on ice

     Heat shock at 42ºC and place on ice

     Incubate with nutrient broth

     Streak plates

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 14

    Transformation of Bacteria

     Play video: Bacterial Transformation

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 15

    How the Calcium Chloride Method Works

     In the presence of

    calcium chloride,

    plasmids are mixed

    with bacteria and

    heat shocked

     Plasmids move into

    the bacteria

    GFP

    Beta-lactamase

    Ampicillin

    Resistance

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 16

    Why Calcium Chloride?

     Helps to neutralize the

    charge on DNA

    molecule, increasing

    probability of that

    molecule moving into

    the cell

    Ca ++

    Ca ++

    O CH 2

    O

    P O

    O

    O Base

    CH 2

    O

    P

    O

    O

    O

    Base

    OH

    Sugar

    Sugar

    O Ca ++

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 17

    What Happens in Each Step?

     Incubate on ice

    – Slows fluid cell membrane

     Heat shock

    – Increases permeability of membranes

     Nutrient broth incubation

    – Allows beta-lactamase expression

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 18

    Electroporation

     Electroporation works by

    – Using electricity to disrupt the bacterial

    wall and membranes

    – Plasmids move in during disruption

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 19

    Other Methods of Moving DNA into Cells

     Biolistics

    – Using microparticles to

    shoot or blast small

    particles coated with DNA

    into cells

    • Plants have a cell wall that

    is difficult to disrupt to

    move DNA into cells

     Transfection

    – Plasmids are placed into

    lipid vesicles

    • The vesicles merge with

    cell membranes and

    deliver DNA into the cells

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 20

    Methods to Select Transformed Cells

     Antibiotic selection

    – When bacteria are plated onto agar that contains antibiotic

    – Bacteria that successfully incorporate a plasmid can grow in

    the presence of antibiotics due to the new enzyme on the

    plasmid

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 21

    Selection of Transformed Cells

     Blue-white screening

    – The β-galactosidase

    enzyme cleaves X-gal

    converting the X-gal into a

    blue color

    – If a gene is successfully

    inserted into the MCS

    (shown in green), then it

    disrupts the cleavage of X-

    gal and will be white in color

    – Antibiotic selection is also

    used to ensure that the

    bacteria were successfully

    transformed initially

  • Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 22

    Selection of