Cloning a Eukaryotic Gene in a Bacterial

Plasmid
Cloning vector
original plasmid in gene cloning
a DNA Molecule that can carry foreign DNA into a host cell and replicate there.
Bacterial plasmids are widely used as cloning vectors
for several reasons:
1. Can be easily isolated from bacteria
2. Can be easily manipulated to form recombinant plasmids by
insertion of foreign DNA in vitro
3. Can be easily introduced into bacterial cells
4. Recombinant bacteria plasmids (foreign DNA they carry multiply
rapidly owing to the high reproductive rate of their host cells.)

Ex. Producing clones of cells using Recombinant plasmids

Studying the B-globin gene in a particular sp. of humming
bird to see if this oxygen-carrying protein is different from
its counterpart in other less metabolically sp.
2 genes: carried by the plasmid
amp^R
• Makes E.coli cells resistant to antibiotic ampicillin
LacZ
• Encodes an enzyme called β-Galactosidase that hydrolyzes the sugar lactose
• Can also hydrolyze a similar synthetic molecule called X-gal to form a blue product.
Site w/in lacZ gene
• Restriction site recognized by restriction enzyme
• only cells with a plasmid will reproduce because only they have the ampR gene.
Example:

Humming bird genes are inserted

into plasmids from E.coli. Only three
plasmids and three hummingbird
DNA fragments are shown, but
millions of copies bird DNA
fragments would be present in the
samples.
Humming- bird DNA fragments

1. Isolate plasmid DNA

from bacterial cells
DNA from
hummingbird cells.
The humming bird
DNA contain the gene
of interest.
hbnnnnj32

2. Cut both DNA

samples with the
same restriction
enzyme, one that
make the single cut
within the lacZ gene
and many cuts within
the hummingbird DNA
3. Mix the cut plasmids and DNA
fragments. Some join by base pairing: add
DNA ligase to seal them together. The
products are recombinant plasmids and
many non-recombinant plasmids

4. Mix the DNA with bacterial cells that

have a mutation in their own lacZ gene.
Some cells take up recombinant plasmid
or other DNA molecule by
transformation.
 RESULTS:
Only a cell who took up a
5. Plate the bacteria on agar
plasmid, which has the amp R
containing ampicillin and X-gal,
gene, will reproduce and form a
incubate until colonies grows.
colony.

 White
 No functional β-
Galactosidase produced
 White color: All DNA
 Will produce functional β-Galactosidase seas from humming
 Enzyme: Blue bird genome
 hydrolyzes the X-gal in the medium
Storing Cloned Genes in DNA Libraries
Genomic library
That is made using bacteria is the collection of the recombinant vector clones
produced by cloning DNA fragments from an entire genome.
That is made using bacteriophages is stored as a collection of clones.
Cut with restriction enzymes into either
small fragments or large fragments
(c) Storing genome libraries

Each clone carries copies of a

particular DNA segment from a
foreign genome, integrated into an
appropriate DNA vector, such as a
plasmid or trimmed down phage
genome.