Gene Cloning

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• Molecular cloning refers to the isolation of individual genes or
other segments of DNA and moving them into an
extrachromosomal DNA from another organism.

• This has two general stages: first, the DNA region or gene of
interest must be isolated and purified; second, this desired
piece of DNA must be inserted into a convenient carrier DNA
molecule or cloning vector

• A variety of DNA molecules may be used as vectors. However,

by far the most popular are plasmids and small viral genomes.

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• Most modern vectors consist of plasmids or viruses
that have been modified to make them more
convenient to use

• The cloning of genes is becoming a very common

procedure

• The term, synthetic biology, refers to the modification

of existing bacteria or other life forms in order to
create a new function, a process that utilizes cloned
genes and vectors.

• The discipline is combining principles of engineering,

biology, and computer analysis to create new biological
solutions to solve many problems.
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 Properties of Cloning Vectors
• In principle, any molecule of DNA, which can replicate itself inside a
cell, could work as a cloning vector.

• For convenience in manipulation, the following factors must be

considered:

• 1. The vector should be a reasonably small and manageable DNA

molecule.

• 2. Moving the vector from one organism to another should be

relatively easy.

• 3. Generating and purifying large amounts of vector DNA should be

straightforward.

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• In addition to these basic requirements, most
vectors have been designed to provide some
convenient means to perform the following:

• 1. A mechanism to select host cells containing

the vector.

• 2. Ability to insert genes into the vector.

• 3. Detect the presence of an inserted gene in

the vector.
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• In practice, bacterial plasmids come closest to these requirements
and are the most widely used vectors.

• These are small, circular pieces of extrachromosomal DNA found

within the cytosol of many different bacteria, archaea, and even
eukaryotes such as yeast and plants.

• They are able to replicate during the cell cycle, and therefore, are
passed down to the daughter cells during cell division.

• Many viruses are also used as vectors, especially when engineering

higher organisms.

• For some special purposes, where very large fragments of DNA are
to be cloned, whole chromosomes are sometimes used as vectors.

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 Plasmid Vectors
• Vectors derived from the small multicopy plasmids of bacteria were
the first to be used and are still the most widespread.

• The ColE1 plasmid of Escherichia coli (E. coli) is a small circular DNA
molecule that forms the basis of many vectors used in molecular
biology.

• The plasmid is named for its colicin E1 gene that makes a toxin that
is released into the environment to kill any surrounding bacteria.

• It exists in up to 40 copies per cell so obtaining plenty of plasmid

DNA is relatively easy and it can be moved from cell to cell by
transformation as described in “Adding Vectors to Host Organisms
with Transformation” .

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• Although the original ColE1 plasmid was once used directly as a vector,
most modern ColE1-based vectors contain a range of artificial
improvements.

• First, the genes for colicin E1 were removed, since these are obviously not
needed.

• Next, an antibiotic resistance gene was added, which makes the host
bacteria resistant to the corresponding antibiotic.

• The most popular antibiotic used for this purpose is ampicillin, a penicillin
derivative.

• The ampicillin resistance gene is known as amp or bla, which refers to its
gene product, beta-lactamase.

• This enzyme inactivates ampicillin found in the bacteria’s environment,

thus protecting its host from death.

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• When a vector that has an antibiotic resistance gene is inside the bacterial
host cell, the resistance gene is transcribed and translated into a protein,
just as any gene in the genome.

• For example, a host bacteria with a vector carrying the bla gene creates
beta-lactamase, and survives because this protein breaks molecules of
ampicillin through hydrolysis, rendering the antibiotic harmless.

• In contrast, the bacteria without beta-lactamase cannot defend

themselves, and the ampicillin degrades the bacteria’s cell wall, which
ultimately kills the bacteria.

• Other resistance proteins work by pumping the antibiotic out of the

bacterial cell or blocking the site in which the antibiotic binds within the
cytoplasm.

• These traits allow the researchers to isolate host cells that harbor the
vector, and separate them from host organisms that do not have a vector.

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 Origin of Replication

• The origin of replication contains the DNA

sequences that are essential for the bacteria
to make copies of the plasmid.

• The pUC series of plasmids can have up to 700

copies of the plasmid in each cell, whereas,
ColE1 plasmids have only 15-20 copies in each
bacteria.

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• The origin also determines whether or not two
different plasmids can be maintained in the same
bacterial cell.

• Plasmids with the same type of origin are

incompatible, and usually one plasmid is rejected
from the bacterial cell.

• So when performing an experiment that requires

two different plasmid constructs in the same
bacterial cell, it is important to choose plasmids
with different origins of replication.
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 Promoters

• Plasmid promoters direct the expression or transcription of the

gene of interest.

• The region includes an RNA polymerase binding site and any

binding sites for regulatory proteins such as transcription factors
that either inhibit or enhance transcription.

• Promoters provide the greatest variability among cloning vectors

because they must be compatible for their host.

• Eukaryotes and prokaryotes have very different promoter

structures, and therefore, cloning vectors must have the correct
elements in order for the host organism to convert the gene into
mRNA.

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• In addition, the promoter offers the researcher a means to
control the expression of their gene of interest.

• The promoter sequences can be changed so that the genes

are only expressed in certain growth conditions, certain
types of day, constantly or constitutively at a low level of
expression, or even constitutively at a very high amount of
expression.

• An inducible promoter only expresses the gene of interest

under defined conditions.

• Some inducible promoters turn on gene transcription when

there is a specific small molecule in the media such as
lactose, galactose, or tetracycline.

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• For example, the pLac promoter from bacteria is off unless
the growth medium has lactose or IPTG (isopropyl -D-
thiogalactoside).

• Repressible promoters turn off gene expression when

certain small molecules such as ethanol or tryptophan are
present.

• The eukaryotic GAL1 and GAL10 promoters have both

repressible and inducible characteristics.

• These promoters are on when galactose is present, but

repressed when glucose is present.

• There are many tissue-specific promoters for specific cell

types, such as neuron, kidney, lung, etc.

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 Adding Inserts to a Vector
• The gene of interest or any DNA fragment of interest is commonly called
the insert.

• The key to getting the insert into the proper location in the vector is to
make the ends of the vector and insert have complementary single-
stranded DNA overhangs.

• There are a variety of methods that accomplish this task.

• The first method, called restriction enzyme cloning, cuts both the DNA
fragment of interest and vector with the same restriction enzyme,
therefore, the ends will have complementary single-stranded overhangs.

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• The cut insert and vector are then mixed with DNA ligase,
which covalently links together DNA strands.

• If the chosen restriction enzyme generates blunt ends,

ligation is more difficult, therefore, T4 ligase is used
because it has the ability to join blunt ends (unlike bacterial
ligase).

• Restriction enzyme cloning is very common, and most

vectors have multiple cloning sites (MCSs) or polylinkers
that have a series of restriction enzyme sites in tandem.

• The restriction enzyme sites found in the MCS are unique,

and only cut the plasmid in that one position.

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 Adding Vectors to Host Organisms With Transformation

• Once an insert has been added to a vector, the new

construct can be inserted into the cytoplasm of a host
organism.

• Since most plasmids are derived from bacteria,

researcher primarily use these constructs in bacterial
hosts.

• The plasmids are easily detected as being present by

conferring a selectable trait to their host, the most
common being an antibiotic resistance gene.
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• Constructs are added to bacterial host cells
using a procedure called transformation.

• First, a tube of bacterial cells bathed in a

solution containing calcium chloride is kept on
ice.

• The calcium ions cause the bacteria to swell,

which makes them competent for taking up
DNA from the environment.
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• After adding the construct to the bacterial cells, the tube is
then switched to a warm water bath, which allows the
constructs to enter the bacterial cell through the small
pores that develop with the temperature change.

• After a brief recovery period at normal temperatures, the

cells are then plated onto nutrient agar to grow overnight.

• The nutrient agar must contain the appropriate antibiotic,

which kills all the bacterial cells that did not take the
construct into their cytoplasm.

• The single bacterial cells that survived the transformation

and have the construct grow into a visible colony overnight.

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 Detecting Inserts in Vectors

• The plasmid itself may be detected by conferring antibiotic

resistance on the host bacteria, but this leaves the question
of whether the insert has combined with the vector before
it was transformed into the host.

• If the cloned gene itself codes for a protein or enzyme that

is easy to detect, there is no problem since the bacteria will
now make this new protein or enzyme.

• In most cases, however, the presence of the inserted DNA

itself must be directly monitored because there is no easy
assay for the gene of interest.

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• One method to screen for plasmid with an insert
is to use a plasmid with two antibiotic resistance
genes.

• One antibiotic resistance gene is used to select

for host cells, which have received the plasmid
vector itself.

• The second is used for the insertion and

detection of cloned DNA.

• The cut site for the restriction enzyme used must

lie within this second antibiotic resistance gene.
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• When the cloned fragment of DNA is inserted,
this antibiotic resistance gene will be disrupted.

• This is referred to as insertional inactivation.

• Consequently, cells that receive a plasmid

without an insert will be resistant to both
antibiotics.

• Those receiving a plasmid with an insert will be

resistant to only the first antibiotic.
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 Reporter Genes
• Genes that are used in genetic analysis because
their products are easy to detect are known as
reporter genes.

• They are often used to report on gene

expression, although they may also be used for
other purposes, such as detecting the location of
a protein or, as here, the presence of a particular
segment of DNA in a cloning vector.
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• One common reporter is lacZ gene encoding β-
galactosidase.

• This enzyme normally splits lactose, a

disaccharide sugar found in milk, into its two
simple sugars, glucose and galactose.

• However, β -galactosidase will also split a wide

range of galactose compounds (i.e., galactosides)
both natural and artificial.

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• The two most commonly used artificial
galactosides are ONPG and X-gal.

• ONPG (o-nitrophenyl galactoside) is split into

o-nitrophenol and galactose.

• The o-nitrophenol is yellow and soluble, so it

is easy to measure quantitatively in solution.

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• X-gal (5-bromo-4-chloro-3-indolyl -D-galactoside)
is split into galactose plus the precursor to an
indigo type dye.

• Oxygen in the air converts the precursor to an

insoluble blue dye that precipitates out at the
location of the lacZ gene.

• Consequently, X-gal is used to monitor β-

galactosidase expression in bacterial colonies on
nutrient agar plates.

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 Blue/White Color Screening
• One widely used method to check for inserts in cloning
vectors uses color screening.

• The most common procedure uses nutrient agar and X-gal

to produce bacterial colonies that change color when an
insert is present within the vector.

• The process, called blue/white screening, uses a vector that

carries the 5’-end of the lacZ gene.

• This truncated gene encodes the alpha fragment of –β-

galactosidase, which consists of the N-terminal region or
first 146 amino acids.
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• A specialized bacterial host strain is required
whose chromosome carries the remaining
portion of the β–galactosidase gene.

• If the plasmid and chromosomal gene

segments are active, they produce two protein
fragments that associate to give an active
enzyme.

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• In order to utilize this unique protein for cloning,
many plasmids are constructed with a polylinker
or MCS located in the middle of the lacZ coding
sequence, very close to the front of the gene
fragment.

• If a foreign segment of DNA is inserted into the

MCS, the alpha fragment of β-galactosidase
becomes disrupted and is not produced.

• The active form of β- galactosidase splits X-gal,

which produces a blue color.
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• Plasmids without a DNA insert will produce β–
galactosidase and the bacterial cell that carries
them will turn blue.

• Plasmids with an insert will be unable to make β-

galactosidase and the cells will stay white.

• In short, a white bacterial colony has the plasmid

with an insert, whereas, the blue bacterial
colonies have a plasmid with NO insert.

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 Types of Cloning Vectors

• There are thousands of different cloning

vectors that are available to researchers.

• This section explains some of the basic

categories of cloning vectors.

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 Moving Genes Among Organisms:
Shuttle Vectors

• Cloned genes are often moved from one organism to

another. This may be done using a shuttle vector.

• As the description implies, this is a vector that can

survive in more than one type of host cell.

• Obviously, the detailed requirements for vectors will

vary depending on the host organism, but the general
ideas are the same.

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 Yeast Artificial Chromosomes
• Analysis of the genomes of higher organisms requires the cloning of
much larger fragments than for bacteria.

• Because eukaryotic genes contain introns they may be hundreds of

kilobases in length, and even the same genes without the introns
can exceed the maximum length for an insert into a plasmid.

• Such large DNA fragments require special vectors.

• The largest capacity vectors derived from bacteriophage can handle

at most 100 kb.

• Consequently, “artificial chromosomes” have been developed to

carry huge lengths of eukaryotic DNA.

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• Huge segments of DNA, up to 2000 kb or 2 million base
pairs, may be carried on yeast artificial chromosomes
(YACs).

• For any replicon to survive in yeast, the vector must

have a yeast specific origin of replication and a
centromere recognition sequence (Cen sequence).

• The YAC has both of these elements. In addition, as

required by all eukaryotic chromosomes, telomere
sequences are present on both ends.

• A yeast cell will treat this structure, although artificial,

as a chromosome.
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 Expression Vectors
• Once a gene has been cloned into a vector, it may or may not be
expressed.

• If both the structural gene and promoter were cloned on the same
segment of DNA, the gene may well be expressed.

• On the other hand, if only the structural gene was cloned then expression
will depend on whether a promoter is provided by the plasmid.

• Vectors that use blue and white screening (discussed earlier) place the
cloned gene under control of the lac promoter, which lies upstream of the
multiple cloning site.

• Often, the objective of cloning a gene is to isolate high levels of the

encoded protein.

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• Purification of proteins has long been complicated
because each protein folds up in an individualized
manner and consequently behaves differently.

• To get around this problem, the target protein is often

tagged with another peptide that is easy to detect
and/or purify.

• This allows purification and manipulation of many

different proteins by the same procedures.

• Tagging is generally done at the genetic level—that is,

an extra segment of DNA that codes for the tag is
inserted next to the DNA coding for the target protein.

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 A DNA Library Is a Collection of Genes
• Gene libraries or DNA libraries are collections of cloned genes that
are big enough to contain at least one copy of every gene from a
particular organism.

• The size of the genes and the organism dictate which vector is used
for holding the inserts.

• The genes of prokaryotes are relatively short, averaging about a

1000 base pairs each.

• In contrast, eukaryotic genes are much longer, largely due to the

presence of introns.

• Different strategies must therefore be followed for prokaryotic and

eukaryotic gene libraries

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• To make a prokaryotic gene library, the complete bacterial
chromosomal DNA is cut with a restriction enzyme and
each of the fragments is inserted into a vector, usually a
simple ColE1-derived plasmid.

• This mixture of vectors containing a different piece of the

bacterial chromosome is transformed into a suitable
bacterial host strain and a large number of colonies, each
containing a single vector plus insert are kept.

• These must then be screened for the gene of interest.

• If the gene has an observable phenotype, this may be used.

• Otherwise, more general methods such as hybridization or

immunological screening are necessary.
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 Cloning Complementary DNA Avoids Introns

• Most eukaryotic genes have intervening

sequences of noncoding DNA (introns)
between the segments of coding sequence
(exons).

• In higher eukaryotes, the introns are often

longer than the exons and the overall length
of the gene is therefore much larger than the
coding sequence.

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• This creates two problems. First, cloning large
segments of DNA is technically difficult; plasmids with
large inserts are often unstable and transform poorly.

• Secondly, bacteria cannot process RNA to remove the

introns and so eukaryotic genes containing introns
cannot be expressed in bacterial cells.

• Using a DNA copy of mRNA, known as complementary

DNA or cDNA, solves both problems since the mRNA
has already been processed by the cell so that all the
introns are removed.

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• To make a cDNA library, the mRNA must be
isolated and used as a template.

• The library generated therefore reflects only

those genes expressed in the particular tissue
under the chosen conditions.

• First, total RNA is extracted from a particular cell

culture, tissue, or specific embryonic stage.

• The mRNA from eukaryotic cells is normally

isolated from the total RNA by taking advantage
of its polyA tail.
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• Since adenine base pairs to thymine or uracil, columns containing
oligo(dT) tracts bound to a solid matrix are used to bind the mRNA
by its polyA tail.

• The mRNA is retained and the other RNA is washed through the
column.

• The mRNA is then released by eluting with a buffer of high ionic

strength that disrupts the H-bonding of the polyA tail to the
oligo(dT).

• A variation of this approach is the use of magnetic beads with

attached oligo(dT) DNA pieces.

• After binding the mRNA, the beads are separated magnetically.

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• To generate cDNA the enzyme reverse
transcriptase, originally found in retroviruses,
is added to the mRNA.

• This enzyme will make a cDNA strand using

the mRNA as template.

• DNA polymerase I is then used to synthesize

the second DNA strand.

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• The resulting double-stranded cDNA
molecules can be isolated and cloned into an
appropriate vector, resulting in a cDNA library.

• Not only is cDNA easier to handle, because

the cloned fragments are much shorter than
the original eukaryotic genes, but also the
cDNA versions of eukaryotic genes can often
be successfully expressed in bacteria.

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