Download Biology 4.13 Plasmids and Vectors

Plasmids and Vectors

Aims:

Must be able to outline what plasmids and
vectors are.

Should be able to explain how plasmids and
vectors are utilised in research.

Could be able to outline the processes for gene
cloning.
Plasmid Vectors

Plasmids are accessory
chromosomes occurring naturally in
bacteria.

Bacterium:
Agrobacterium
tumefaciens
In nature, plasmids are usually
transferred between closely related
microbes by cell-to-cell contact (a
process called conjugation).
Plasmid

Simple chemical treatments can
Plant infected
by bacterium
with foreign
gene
make mammalian cells, yeast cells
and some bacterial cells that do not
naturally transfer DNA, able to take
up external DNA.
Plasmid with
foreign gene
Recombinant DNA Plasmid

The fragments of DNA are joined together by the enzyme DNA
ligase, producing a molecule of recombinant DNA.

These combined techniques of using restriction enzymes and
ligation are the basic tools of genetic engineering.
DNA ligase
Detail of Restriction Site
Recombinant
Plasmid DNA
Fragments
linked
permanently by
DNA ligase
No break in
DNA molecule
The fragments are able
to join together under
the influence of DNA
ligase.
Using
Plasmids

Plasmid
vectors, found in
bacteria, are
prepared for
cloning
Preparation of the Clone
Human cell
Human gene
DNA in
chromosome
Escherichia coli
bacterial cell
Sticky
end
A gene of interest (DNA
fragment) is isolated from
human tissue cells
Plasmid
Chromosome
Sticky
end
Restriction enzyme
recognition sequence
Human DNA and plasmid are
treated with the same restriction
enzyme to produce identical
sticky ends
Plasmid
vector
Ampicillinresistance
gene
Tetracyclineresistance gene
Gene
disrupted
Sticky
ends
An appropriate
plasmid vector is
isolated from a
bacterial cell
Restriction enzyme cuts the plasmid DNA
at its single recognition sequence,
disrupting the tetracycline resistance gene
following steps
1-6:
Mix the DNAs together and add
the enzyme DNA ligase to bond Human gene
the sticky ends
Recombinant plasmid is introduced
into a bacterial cell by simply adding
the DNA to a bacterial culture where
some bacteria take up the plasmid
from solution
Recombinant
DNA
molecule
Activity

Answer the questions on p237/8 in the
Biozone books.
Gene Cloning – Making Copies

Gene cloning = Process of making large quantities of a desired piece of
DNA after isolation.

Biologists obtain genes for cloning from two main sources:

DNA isolated directly from an organism.

Complementary DNA made in the laboratory from messenger RNA (mRNA)
templates.

Allows for an unlimited number of copies of a gene to be produced for
analysis or for production of a protein product.

Methods have been developed to insert a DNA fragment of interest (e.g. a
segment of human DNA) into the DNA of a vector, resulting in a
recombinant DNA molecule or molecular clone.

Large quantities of the desired gene can be obtained if the recombinant
DNA is allowed to replicate in an appropriate host.
Vectors for Gene Cloning

Vector = self-replicating DNA molecule (e.g. plasmid or viral DNA) used to
transmit a gene from one organism into another.

All vectors must have the following properties:


Be able to replicate inside their host organism.

Have one or more sites at which a restriction enzyme can cut.

Have some kind of genetic marker that allows them to be easily identified.
Organisms such as bacteria, viruses and yeasts have DNA which behaves
in this way.
Cloning
the Gene

The gene cloning
process (making
Bacterium
multiple copies of the
Bacteria containing the
recombinant plasmid are
spread onto an agar
plate containing
ampicillin.
Dish A
human gene) occurs
Human gene
Recombinant plasmid
when the bacterium
with the recombinant
All colonies look identical but
only some have the plasmid
with the human gene.
plasmid is allowed to
Agar containing ampicillin allows
only bacterial colonies with the
appropriate plasmid to grow.
reproduce.

Filter
paper
Dish B
Filter paper is pressed against the
agar plate thereby transferring
colonies of bacteria to the paper.
Colonies of bacteria
that carry the
recombinant plasmid
The filter paper is pressed against agar that
contains tetracycline. Those colonies that
grow do not have the human gene
disrupting the tetracycline resistance gene.
Colonies with the human gene
can be located according to their
position on the original dish A.
can be identified by
differential response to
antibiotics