Download Transformation and Cloning

Transformation and Cloning
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Transformation
• Remember that a gene is a piece of DNA.
• It provides the instructions (codes) for a protein that gives an
organism a particular trait.
Genetic transformation
• This is the uptake of naked DNA from the environment.
• Can induce this to happen in a laboratory situation – inserting
the DNA you want the microbe to express.
• Uptake of new DNA by microorganism will provide cell with new
characteristics.
• This technique used widely in biotechnology.
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In the laboratory situation
• From a population of 1000 or more treated cells only
one or two may pick up foreign DNA.
• Those which do are said to be transformed.
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Bacterial transformation
Antibiotic-sensitive bacterial cell
Calcium chloride treatment permeablises cell wall
Plasmid DNA
Transformed bacterial cell
Transformed bacteria selected by growing on solid
media containing appropriate antibiotic
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Recipients for foreign DNA
• Bacterium E. coli and the yeast S. cerivisiae are
widely used as recipients for foreign DNA.
• Both are unicellular and quick growing.
• Ideal for large-scale production of proteins such as
industrial enzymes.
Discuss the advantages and disadvantages of using
E. coli and S. cerivisiae.
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E. coli advantages
• Several different types of plasmid can be used to
introduce foreign DNA.
• Foreign DNA can account for up to 60% of its total
protein production.
• Fast growing.
• Easy to transform.
• Easy to manipulate.
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E. coli disadvantages
• Does not carry out post-translational modifications to
proteins.
• These modifications are necessary for protein
function in, for example, human cells.
• Bacterium occurs naturally in the intestines of
humans and under certain circumstances can cause
disease.
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S. cerivisiae advantages
• Rarely a human pathogen.
• Genes organised, expressed and controlled in ways
that are similar to human genes.
• Carries out post-translational modifications, eg
addition of sugar residues, which are a common
feature of human proteins.
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S. cerivisiae disadvantages
• Difficult to transform.
• Yields less protein than bacteria.
• Plasmids easily lost from yeast – no selective
advantage to yeast.
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Cloning vectors
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Cloning vectors
• In gene cloning, once recombinant DNA (rDNA) has
been constructed it is introduced into a host.
• In the host, rDNA has to be:
maintained
replicated
passed from one generation to another.
• This is achieved by introducing rDNA into a cell
on a DNA vehicle called a cloning vector – most
commonly used are plasmid cloning vectors.
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Plasmids
• Extra-chromosomal
DNA found in bacteria.
• Loops of doublestranded DNA.
• Some present in
multiple copies.
• Independently replicate
inside bacteria.
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Artificial plasmids
• Artificial plasmids have been
genetically engineered for
the purpose of cloning.
• Used as vectors, ie to
transfer DNA from one cell
to another.
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Plasmids
• Purpose of a vector is to carry DNA into a cell.
• Foreign DNA must be cut with same enzyme as
plasmid so the ‘ends’ are compatible – plasmid and
foreign DNA are then joined by ligase enzyme.
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Features of plasmids
Plasmids also contain a selectable marker.
• Usually an antibiotic resistance gene:
– required for maintenance of plasmid in the cell
– advantageous for bacteria to keep the plasmid (can
then grow in presence of antibiotic).
• Commonly used selectable markers are ampicillin,
neomycin and chloramphenicol.
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Example of controlling expression using an inducer
Gene for green fluorescent protein from jellyfish can
be inserted into various other cell types.
Gene has been combined in a plasmid along with
control elements from arabinose operon ( ie usually
regulates synthesis of enzymes needed to metabolise
the sugar arabinose).
Can turn expression of the ‘glowing protein’ on by adding
arabinose to the growth medium.
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Alba the pGLO bunny!
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Steps in transforming bacteria
Bacteria incubated with plasmid (with appropriate promoter
and antibiotic resistance gene).
Bacteria plated out onto agar plates containing antibiotic.
Only those bacteria that have taken up plasmid will be able to
grow on agar plus antibiotic.
Transformed bacteria can then be isolated and grown in bulk
with appropriate antibiotic. Bacteria multiply to produce
genetically identical offspring – clones.
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