CHOICE OF CLONING VECTORS

Vectors must be small molecules for convenient manipulation.

They must be capable of prolific replication in a living cell in order to amplify

the inserted donor fragment.

They must also have convenient restriction sites at which the DNA to be
cloned may be inserted. Ideally, the restriction site should be present only once in the
vector because then restriction fragments of donor DNA will insert only at that one
location in the vector.

It is also important that there be a way to identify and recover the recombinant molecule
quickly.

Numerous cloning vectors are in current use, suitable for different sizes of DNA insert or
for different uses of the clone.
 Good plasmid cloning vehicles share a number of desirable features

An ideal cloning vehicle would have the following three properties:

• low molecular weight;

• ability to confer readily selectable phenotypic traits on host cells;
• single sites for a large number of restriction endonucleases, preferably
in genes with a readily scorable phenotype.

Ability to replicate within the host cell, so that numerous copies of the
recombinant DNA molecule can be produced and passed to daughter
cells.
A DNA fragment of interest is covalently joined to a DNA vector.

The essential feature of a vector is that it can replicate autonomously in an appropriate

host.

Plasmids (naturally occurring circles of DNA that act as accessory chromosomes in

bacteria) and bacteriophage lambda ( phage), a virus, are choice vectors for cloning in E.
coli.

Plasmids are widely used as cloning vehicles. They are stably inherited in an
extrachromosomal state.

Most plasmids exist as double-stranded circular DNA molecules.

The plasmids used as vectors carry genes for drug resistance.

These drug-resistance genes provide a convenient way to select for cells transformed
by plasmids: those cells still alive after exposure to the drug must carry the plasmid
vectors containing the DNA insert.
 Plasmids are also an efficient means of amplifying cloned DNA because there are many
copies per cell, as many as several hundred for some plasmids.

General-purpose cloning vectors. Cloning of foreign DNA fragments in general-purpose

cloning vectors (e.g., pBR322 [11]) selectively inactivates one of the markers (insertional
inactivation) or derepresses a silent marker (positive selection) so as to differentiate the
recombinants from the native phenotype of the vector.

Expression vectors. In expression vectors (e.g., pUC18 [123]), DNA to be cloned and
expressed is inserted downstream of a strong promoter present in the vector.
The vector can be prepared for accepting a new DNA fragment by cleaving it at a single
specific site with a restriction enzyme. For example, the plasmid pSC101, a 9.9-kb double-
helical circular DNA molecule, is split at a unique site by the EcoRI restriction enzyme.

The staggered cuts made by this enzyme produce complementary single-stranded ends,
which have specific affinity for each other and hence are known as cohesive or sticky ends.

Any DNA fragment can be inserted into this plasmid if it has the same cohesive ends.

Such a fragment can be prepared from a larger piece of DNA by using the same restriction
enzyme as was used to open the plasmid DNA
Attaching donor and
vector DNA
Most commonly, both
donor and vector DNA are
digested
by a restriction enzyme
that produces
complementary
sticky ends and are then
mixed in a test tube to
allow the sticky ends of
vector and donor DNA to
bind
to each other and form
recombinant molecules.
 Plasmids can also be categorized on
the basis of their being maintained as
multiple copies per cell (relaxed
plasmids) or as a limited number of
copies per cell (stringent plasmids).

After a piece of foreign DNA is inserted into a

vector, the resulting chimeric molecules have to
be transformed into a suitable recipient.

Since the efficiency of transformation is so low,

it is essential that the chimeras have some
readily scorable phenotype.

Usually this results from some gene, e.g.

antibiotic resistance, carried on the vector, but
could also be produced by a gene carried on
the inserted DNA.
How amplification works. Restriction-enzyme treatment of donor DNA and vector allows the
insertion of single fragments into vectors. A single vector enters a bacterial host, where replication and
cell division result in a large number of copies of the donor fragment.
• The judicious choice of markers on cloning vectors can greatly simplify the selection
and analysis of recombinant clones.

• A key step in any cloning procedure is the selection of transformants carrying the
desired recombinant plasmid.

• Because transformation efficiencies are so low it is essential to be able to select

positively those rare cells that have been transformed.

• The commonest selectable markers are ones encoding resistance to antibiotics such as
ampicillin (ApR), chloramphenicol (CmR), tetracycline (TcR), streptomycin (SmR), and
kanamycin (KmR).

• Another type of positive selection is reversal of auxotrophy. For example, if the

hisB+gene is cloned in a vector then it is easy to select recombinants by transforming a
hisB auxotroph and growing it in a medium lacking histidine.

• Reporter genes are ones whose phenotype can be discerned by visual examination of
colonies growing on a plate and/or ones that can be used to measure levels of gene
expression. In terms of analysis of recombinants, the most widely used reporter
gene is the lacZ gene encoding β-galactosidase.
The pUC vectors also incorporate a DNA sequence that permits rapid visual
detection of an insert.

The MCS is inserted into the IacZ sequence,which encodes the promoter and the a-
peptide of ~-galactosidase.

The insertion of the MCS into the lacZ' fragment does not affect the abilityof the a-
peptide to mediate complementation, but cloning DNA fragments into the MCS does.

Therefore,recombinants can be detected by blue/white screening on growth medium

containing Xgal.
 Prokaryotic Gene Regulation
Despite the simplicity of their form, bacteria have a fundamental need to regulate the
expression of their genes.

One of the main reasons is that they are nutritional opportunists.

Consider how bacteria obtain the many important compounds, such as sugars, amino
acids, and nucleotides, needed for metabolism.

Bacteria swim in a sea of potential nutrients. They can either acquire these
compounds from the environment or synthesize them by enzymatic pathways.

Synthesizing the necessary enzymes for these pathways expends energy and cellular
resources; so, given the choice, bacteria will take compounds from the environment
instead.

To be economical, they will synthesize the enzymes necessary to produce these

compounds only when there is no other option—in other words, when these
compounds are unavailable in their local environment.
Bacteria have evolved regulatory systems that couple the expression of gene products to
sensor systems that detect the relevant compound in a bacterium’s local environment.

The regulation of enzymes taking part in sugar metabolism provides an example.

Sugar molecules can be oxidized to provide energy or they can be used as building blocks
for a great range of organic compounds.

However, there are many different types of sugar that bacteria could use, including lactose,
glucose, galactose, and xylose.

First, a different import protein is required to allow each of these sugars to enter the cell.

Further, a different set of enzymes is required to process each of the sugars.

If a cell were to simultaneously synthesize all the enzymes that it might possibly need, the
cell would expend much more energy and materials to produce the enzymes than it could
ever derive from breaking down prospective carbon sources.
Cells need mechanisms that fulfill two criteria:

1. They must be able to recognize environmental conditions in which they should

activate or repress transcription of the relevant genes.

2. They must be able to toggle on or off the transcription of each specific gene or
group of genes.

Bacteria have a simple general mechanism for coordinating the regulation of genes
encoding products that participate in a set of related processes: these genes are
clustered on the chromosome and are transcribed together.

Many prokaryotic mRNAs are polycistronic— multiple genes on a single transcript—and

the single promoter that initiates transcription of the cluster is the site of regulation for
expression of all the genes in the cluster.

The gene cluster and promoter, plus additional sequences that function together in
regulation, are called an operon
 Lac Operon:
Many of the principles of prokaryotic gene expression were first defined by studies of
lactose metabolism in E. coli, which can use lactose as its sole carbon source.

In 1960, François Jacob and Jacques Monod published a short paper in the
Proceedings of the French Academy of Sciences that described how two adjacent genes
involved in lactose metabolism were coordinately regulatedby a genetic element located
at one end of the gene cluster.

The genes were those for b-galactosidase, which cleaves lactose to galactose and
glucose, and galactoside permease, which transports lactose into the cell .

The terms “operon” and “operator” were first introduced in this paper.
 Lac Operon:
Many prokaryotic mRNAs are polycistronic— multiple genes on a single transcript—and
the single promoter that initiates transcription of the cluster is the site of regulation for
expression of all the genes in the cluster.

The gene cluster and promoter, plus additional sequences that function together in
regulation, are called an operon.

The lactose (lac) operon includes the genes for b-galactosidase (Z), galactoside
permease (Y), and thiogalactoside transacetylase (A).

The last of these enzymes appears to modify toxic galactosides to facilitate their removal
from the cell.
Two types of DNA–protein interactions are required for regulated transcription. Both take
place near the site at which gene transcription begins.

One of these DNA–protein interactions determines where transcription begins. The DNA
that participates in this interaction is a DNA segment called the promoter, and the protein
that binds to this site is RNA polymerase.

When RNA polymerase binds to the promoter DNA, transcription can initiate a few bases
away from the promoter site. Every gene must have a promoter or it cannot be
transcribed.
The basics of prokaryotic transcriptional regulation

The other type of DNA–protein interaction decides whether promoter-driven transcription

takes place. DNA segments near the promoter serve as binding sites for regulatory
proteins called activators and repressors.

In bacteria, most of these sites are termed operators.

For some genes, an activator protein must bind to its target DNA site as a necessary
prerequisite for transcription to begin.

For some genes, an activator protein must bind to its target DNA site as a necessary
prerequisite for transcription to begin.

Such instances are sometimes referred to as positive regulation because the presence
of the bound protein is required for transcription.

For other genes, a repressor protein must be prevented from binding to its target site as a
necessary prerequisite for transcription to begin. Such cases are sometimes termed
negative regulation because the absence of the bound repressor allows transcription to
begin.
https://www.youtube.com/watch?v=sc9pAk0blgo