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BIO 103
EXPERIMENT 5
Plasmid Transformation
Transformation
 is the genetic alteration of a cell resulting from the introduction,
uptake and expression of foreign genetic material (DNA or RNA).
 is a common technique in molecular biology.
 Historically,
o 1865 - Gregor Mendel develops a model of genetic heredity
by the passing down of traits.
o 1928 - Frederick Griffith transforms nonpathogenic
pneumococcus bacteria into a virulent variety by immersing
them in heat- killed pathogenic material.
o 1944 - Oswald Avery, Colin MacLeod, and Maclyn McCarty
announce that they have discovered the transforming
factor - DNA.
 in bacteria, yeasts, fungi, plants and animals.
Bacterial Transformation
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 Transformation refers to a genetic change brought about by
taking up and expressing DNA.
 Competence refers to the state of being able to take up DNA.
Two different forms of competence should be distinguished,
natural and artificial.
o Natural competence : Transformation occurs only in
bacterial species capable of natural competence. Such
species carry sets of genes specifying machinery for
bringing DNA across the cell's membrane or membranes.
o Artificial competence: This is not encoded in the cell's
genes. Instead it is a laboratory procedure in which cells are
passively made permeable to DNA, using conditions that do
not normally occur in nature. These procedures are
comparatively easy and simple, and can be used to
genetically engineer bacteria.
Artificial Competence
 Chilling cells in the presence of divalent cations such as CaCl2
prepares the cell walls to become permeable to plasmid DNA.
Cells are incubated with the DNA and then briefly heat shocked
(42°C for 30-120 seconds), which causes the DNA to enter the
cell. This method works well for circular plasmid DNAs but not
for linear molecules such as fragments of chromosomal DNA.
 Electroporation is another way to make holes in cells, by briefly
shocking them with an electric field of 100-200V. Now plasmid
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DNA can enter the cell through these holes. Natural membranerepair mechanisms will close these holes afterwards.
What are plasmids?
 is a DNA molecule separate from the chromosomal DNA and
capable of autonomous replication.
 is typically circular and double-stranded. It usually occurs in
bacteria, sometimes in eukaryotic organisms (e.g., the 2micrometre-ring in Saccharomyces cerevisiae).
 Size of plasmids varies from 1 to over 400 kbp.
 There may be one copy, for large plasmids, to hundreds of copies
of the same plasmid in a single cell, or even thousands of copies,
for certain artificial plasmids selected for high copy number
(such as the pUC series of plasmids).
Antibiotic Resistance and Origin of Replication
 Plasmids often contain genes that confer a selective advantage to
the bacterium harboring them, such as the ability to make the
bacterium antibiotic resistant.
 Every plasmid contains at least one DNA sequence that serves as
an origin of replication, or ori (a starting point for DNA
replication), which enables the plasmid DNA to be duplicated
independently from the chromosomal DNA.
Vectors
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 Plasmids used in genetic engineering are called vectors.
 They are used to transfer genes from one organism to another
and typically contain a genetic marker conferring a phenotype
that can be selected for or against.
 Most also contain a polylinker or multiple cloning site (MCS),
which is a short region containing several commonly used
restriction sites allowing the easy insertion of DNA fragments at
this location.
Restriction Enzymes
 enzymes that cut double-stranded DNA, on each strand, through
the sugar-phosphate backbones.
 are believed to be a mechanism evolved by bacteria to resist viral
attack and to help in the removal of viral sequences.
 The 1978 Nobel Prize in Medicine was awarded to D. Nathans and
H. Smith for the discovery of restriction enzymes, leading to the
development of recombinant DNA technology; (eg.human insulin
for diabetics).
Applications
Plasmids serve as important tools in genetics and biochemistry labs,
where they are commonly used to multiply (make many copies of) or
express particular genes.
 The gene to be replicated is inserted into copies of a plasmid
which contains genes that make cells resistant to particular antibiotics.
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 Next, the plasmids are inserted into bacteria by transformation.
 The bacteria are exposed to the particular antibiotics. Only
bacteria which take up copies of the plasmid survive the
antibiotic, since the plasmid makes them resistant.
 Now these bacteria can be grown in large amounts, harvested and
lysed to isolate the plasmid of interest.
α-Complementation
 The lac operon is an operon required for the transport and
metabolism of lactose in Escherichia Coli and some other enteric
bacteria.
 The lac operon consists of three structural genes, a promoter, an
operator, and a terminator. The three structural genes are: lacZ,
lacY, and lacA.
 lacZ encodes β-galactosidase, an intracellular enzyme that
cleaves the disaccharide lactose into glucose and galactose.
 The vector contains the regulatory portion of the lac operon and
codes for the first 146 amino acids of the β-galactosidase
enzyme.
 The host cell contains the missing carboxyl portion of the βgalactosidase protein but lacks the region in the vector.
 Thus, in host cells that contain intact vector, the two portions of
the β-galactosidase protein can complement to form a functional
enzyme.
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Cloning genes as a technique to see whether plasmids have acquired
foreign genetic material...
If X-gal and IPTG are contained within an agar medium on a culture
plate, you can easily distinguish which colonies have a functional LacZ
gene.
 IPTG (Isopropyl-β-D-thio-galactoside) is frequently used as an
inducer of the lac operon for physiological work. It binds to
repressor and inactivates it, but is not a substrate for βgalactosidase.
 X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) is
cleaved by β-galactosidase yielding galactose and 5-bromo-4chloro-3-hydroxyindole, which then is oxidized into 5,5'-dibromo4,4'-dichloro-indigo, an insoluble blue product.
=> Thus,
o E. Coli bacteria which contain the intact plasmid will produce βgalactosidase and turn in blue color.
o E. Coli bacteria which contain the plasmid that acquired
foreign DNA (this will disrupt the lacZ gene) will be unable to
produce β-galactosidase and no blue color will appear.
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