ON

BACTERIAL PLASMID AS VECTORS

SUBJECT:- CONCEPTS IN BIOTECHNOLOGY

SUBJECT CODE:-BTC-012

SUBMITTED TO:- SUBMITTED BY:-

Mr. HARSH KUMAR NISHU KUMAR BANSAL

(Lect. In BIOTECHNOLOGY) B.Tech(Bio Tech)- 3rd sem

Section- A

ROLL NO. R108A05

Reg. NO.1040070148

LOVELY PROFESSIONAL UNIVERSITY

 PREFACE

Genetic engineering is a new technology that combines genes from totally unrelated
species, in combinations not possible using conventional breeding methods. Genes from
an animal, say, a fish, can be put into a plant, a strawberry for instance. In fact this is an
actual example of an attempt to "improve" strawberry plants. The fish gene is supposed to
make the strawberries more resistant to frost by causing the strawberry plant to produce a
form of antifreeze which the fish normally produces to endure cold ocean conditions.

Over 60 percent of all processed foods purchased by U.S. consumers are manufactured
with Genetic engineered ingredients. Some corn and potatoes have even been genetically
engineered to contain a gene from Bt bacteria which causes every cell of the plants to
produce an insecticidal toxin. Yet there is no labeling of these or any GE foods as being
genetically engineered, because the U.S. Food and Drug Administration (FDA) considers
the genetically engineered organisms (GEOs) from which these foods are made to be
"substantially equivalent" to the non-genetically engineered plant from which the GEOs
are derived.

The primary goal of this term parer has been to provide useful information about genetic
engineering in a simple and illustrative language . The organization of the material Is
largely traditional and the order in which the content arranged, is a matter of personal
choice. I hope you will enjoy reading.

 Thank you
 ACKNOWLEDGEMENT

I would like to express my gratitude to all those who gave me the possibility to complete
this term paper. I want to thank the Department of BIOTECHNOLOGY of LOVELY
PROFESSIONAL UNIVERSITY for giving me permission to commence this Term
paper, to do the necessary research work and to use departmental data. I have furthermore
to thank to Mr. HARSH KUMAR , Lect. in BlOTECHNOLOGY who gave and
confirmed this permission and encouraged me to go ahead with my term paper.

I am again deeply indebted to my supervisor Lect. HARSH SIR whose help, stimulating
suggestions and encouragement helped me in all the time of research for and writing of
this term paper.

 Thank you
 Contents

Introduction
Plasmid
Vectors
Properties of a good vector
 A cloning site
 An origin of replication
A selectable marker gene

How to Clone DNA Fragments

 Fragmentation
 Ligation
 Transfection
 Screening and Selection
 Conformation

Types of plasmids
 Plasmid DNA extraction
 Conformations
Inserting a DNA Sample into a Plasmid
pBR322: description & restriction map
References
 INTRODUCTION

Biotechnology has to do with the manipulation of organisms to get useful products. One
of the basis of biotechnology is genetic transformation. Genetic transformation
occurs when DNA is taken in and expressed by a cell from a living organism. Three
basic thing are needed to perform genetic transformation: a host, a vector, and a
method to select and isolate the transformed organisms. The host is the organism that
will take in the DNA. The vector is the means of transporting the DNA into the host.
A common method for selecting the transformed organisms is to introduce them to
an environment containing what they are resistant to (once they transform) because
only the transformed organisms will be able to survive in that environment.
In this term paper we will use bacterial plasmid as vector. Bacterial cells may contain
a number of plasmids which have different and varied functions such as funtions for
colicin resistance, symbiosis, nitrogen fixation, and tumour induction in plants.
Among the first to be discovered -- soon after the discovery of the F plasmid -- were
the resistance transfer factors, which have been extensively modified and adapted
over the years since to become the modern cloning plasmids.

Bacterial Transformation is a very basic technique that is used on a daily basis in a

molecular biological laboratory. The purpose of this technique is to introduce a foreign
plasmid into a bacteria and to use that bacteria to amplify the plasmid in order to make
large quantities of it. This is based on the natural function of a plasmid: to transfer
genetic information vital to the survival of the bacteria.
Plasmid:-

Illustration of a bacterium with plasmids enclosed showing chromosomal DNA and

plasmids.

A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA

which is capable of replicating independently of the chromosomal DNA. In many cases,
it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are
sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces
cerevisiae).

Plasmid size varies from 1 to over 200 kilobase pairs (kbp). The number of identical
plasmids within a single cell can range anywhere from one to even thousands under some
circumstances. Plasmids can be considered to be part of the mobilome, since they are
often associated with conjugation, a mechanism of horizontal gene transfer.

The term plasmid was first introduced by the American molecular biologist Joshua
Lederberg in 1952.

Plasmids can be considered to be independent life-forms similar to viruses, since both are
capable of autonomous replication in suitable (host) environments. However the plasmid-
host relationship tends to be more symbiotic than parasitic (although this can also occur
for viruses, for example with endoviruses) since plasmids can endow their hosts with
useful packages of DNA to assist mutual survival in times of severe stress. For example,
plasmids can convey antibiotic resistance to host bacteria, who may then survive along
with their life-saving guests who are carried along into future host generations.

Vectors:-
There are two types of plasmid integration into a host bacteria: Non-integrating plasmids
replicate as with the top instance; whereas episomes, the lower example, integrate into
the host chromosome.

Plasmids used in genetic engineering are called vectors. Plasmids serve as important tools
in genetics and biotechnology labs, where they are commonly used to multiply (make
many copies of) or express particular genes. Many plasmids are commercially available
for such uses.

The gene to be replicated is inserted into copies of a plasmid containing genes that make
cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker),
which is a short region containing several commonly used restriction sites allowing the
easy insertion of DNA fragments at this location. Next, the plasmids are inserted into
bacteria by a process called transformation. Then, 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. In particular, the protecting genes are
expressed (used to make a protein) and the expressed protein breaks down the antibiotics.
In this way the antibiotics act as a filter to select only the modified bacteria. Now these
bacteria can be grown in large amounts, harvested and lysed (often using the alkaline
lysis method) to isolate the plasmid of interest.

Another major use of plasmids is to make large amounts of proteins. In this case,
researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the
bacteria produces proteins to confer its antibiotic resistance, it can also be induced to
produce large amounts of proteins from the inserted gene. This is a cheap and easy way
of mass-producing a gene or the protein it then codes for, for example, insulin or even
antibiotics.

However, a plasmid can only contain inserts of about 1-10 kbp. To clone longer lengths
of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial
chromosomes or yeast artificial chromosomes could be used.
Properties of a good vector

A plasmid is an extra-chromosomal element, often a circular DNA. The plasmids we will

use in this class typically have three important elements:

• A cloning site (a place to insert foreign DNAs)

• An origin of replication
• A selectable marker gene (e.g. resistance to ampicillin)

1.Cloning sites:

A cloning site is not required at all, but it sure is nice to have! What I mean by "cloning
site" is a place where the DNA can be digested by specific restriction enzymes - a
point of entry or analysis for genetic engineering work. This is a matter we will be
discussing in great detail at a later point. For now, think of the following example:
Suppose you are really thirsty and you buy a can of beer. Does it occur to you that
one end of the can (the "top") is designed so that you can open it easily? If you
bought a can of beer with two bottom ends and no top, you would have a hard time
drinking it! It's the same way with plasmids. You can have a plasmid with lots of
terrific features, but you might lack an easy way of "getting it open" with restriction
enzymes. All this talk is making me thirsty, and now I believe I'll have that drink!

2.Origins of replication:

Since a plasmid is (by definition) an extrachromosomal element, it cannot make use of

any origin of DNA replication in a chromosome. That is, DNA synthesis within
(i.e. copying of) a plasmid depends on its having an origin of DNA synthesis of its
own. Obviously, if a plasmid couldn't be copied, it would be rapidly diluted out in a
population of dividing cells because it couldn't be passed on to daughter cells.

3.Selectable markers:
A selectable marker is not actually a required element of a plasmid, but it makes it
possible for us to maintain stocks of cells that contain the plasmid uniformly.
Sometimes, carrying a plasmid puts a cell at a selective disadvantage compared to
its plasmid-free neighbors, so the cells with plasmids grow more slowly. Cells that
happen to "kick out" their plasmid during division may be "rewarded" by having a
higher rate of growth, and so these plasmid-free (sometimes referred to as "cured")
cells may take over a population. If a plasmid contains a gene that the cell needs to
survive (for example, a gene encoding an enzyme that destroys an antibiotic), then
cells that happen to kick out a plasmid are "punished" (by subsequent death) rather
than "rewarded" (as in the previous scenario). That selective pressure helps to
maintain a plasmid in a population

How to Clone DNA Fragments –

Diagram of the cloning vector puc 19 (from dwb.unl.edu/.../genome/

moltec/vectors/puc19.htm).
 Fragmentation - Initially, the DNA of interest needs to be fragmented to
provide a relevant DNA segment of suitable size. Preparation of DNA fragments for
cloning is frequently achieved by means of PCR, but it may also be accomplished by
restriction enzyme digestion and sometimes fractionation by gel electrophoresis.
 Ligation - Subsequently, a ligation procedure is employed whereby the
amplified fragment is inserted into a vector. The vector (which is frequently circular) is
linearised by means of restriction enzymes, and incubated with the fragment of interest
under appropriate conditions with an enzyme called DNA ligase.
 Transfection - Following ligation the vector with the insert of interest is
transfected into cells. Most commonly electroporation is employed, although a number
of alternative techniques are available, such as chemical sensitization of cells.
 Screening and Selection - Finally, the transfected cells are cultured. As the
aforementioned procedures are of particularly low efficiency, there is need to identify
the cell colonies that have been successfully transfected with the vector construct
containing the desired insertion sequence. Modern cloning vectors include selectable
antibiotic resistance markers, which allow only cells in which the vector has been
transfected, to grow. Additionally, the cloning vectors may contain color selection
markers which provide blue/white screening on X-gal medium.
 Conformation - Nevertheless, these selection steps do not absolutely
guarantee that the DNA insert is present in the cells obtained. Further investigation of
the resulting colonies is required to confirm that cloning was successful. This may be
accomplished by means of PCR, restriction fragment analysis and DNA sequencing

Types of plasmids:- It based upon two things:-

 On basis of conjugation:-
Overview of Bacterial conjugation

One way of grouping plasmids is by their ability to transfer to other bacteria. Conjugative
plasmids contain so-called tra-genes, which perform the complex process of conjugation,
the transfer of plasmids to another bacterium (Fig. 4). Non-conjugative plasmids are
incapable of initiating conjugation, hence they can only be transferred with the assistance
of conjugative plasmids, by 'accident'. An intermediate class of plasmids are mobilizable,
and carry only a subset of the genes required for transfer. They can 'parasitize' a
conjugative plasmid, transferring at high frequency only in its presence. Plasmids are
now being used to manipulate DNA and may possibly be a tool for curing many diseases.
It is possible for plasmids of different types to coexist in a single cell. Seven different
plasmids have been found in E. coli. But related plasmids are often incompatible, in the
sense that only one of them survives in the cell line, due to the regulation of vital plasmid
functions. Therefore, plasmids can be assigned into compatibility groups.

 On basis of function:-

Another way to classify plasmids is by function. There are five main classes:

• Fertility-F-plasmids, which contain tra-genes. They are capable of conjugation

(transfer of genetic material between bacteria which are touching).
• Resistance-(R)plasmids, which contain genes that can build a resistance against
antibiotics or poisons. Historically known as R-factors, before the nature of
plasmids was understood.
• Col-plasmids, which contain genes that code for (determine the production of)
bacteriocins, proteins that can kill other bacteria.
• Degradative plasmids, which enable the digestion of unusual substances, e.g.,
toluene or salicylic acid.
• Virulence plasmids, which turn the bacterium into a pathogen (one that causes
disease).

Plasmids can belong to more than one of these functional groups.

Plasmids that exist only as one or a few copies in each bacterium are, upon cell division,
in danger of being lost in one of the segregating bacteria. Such single-copy plasmids have
systems which attempt to actively distribute a copy to both daughter cells.

Some plasmids include an addiction system or "postsegregational killing system (PSK)",

such as the hok/sok (host killing/suppressor of killing) system of plasmid R1 in
Escherichia coli. They produce both a long-lived poison and a short-lived antidote.
Daughter cells that retain a copy of the plasmid survive, while a daughter cell that fails to
inherit the plasmid dies or suffers a reduced growth-rate because of the lingering poison
from the parent cell.
Plasmid DNA extraction:-

As alluded to above, plasmids are often used to purify a specific sequence, since they can
easily be purified away from the rest of the genome. For their use as vectors, and for
molecular cloning, plasmids often need to be isolated.

There are several methods to isolate plasmid DNA from bacteria, the archetypes of which
are the miniprep and the maxiprep/bulkprep. The former can be used to quickly find out
whether the plasmid is correct in any of several bacterial clones. The yield is a small
amount of impure plasmid DNA, which is sufficient for analysis by restriction digest and
for some cloning techniques.

In the latter, much larger volumes of bacterial suspension are grown from which a maxi-
prep can be performed. Essentially this is a scaled-up miniprep followed by additional
purification. This results in relatively large amounts (several micrograms) of very pure
plasmid DNA.

In recent times many commercial kits have been created to perform plasmid extraction at
various scales, purity and levels of automation. Commercial services can prepare plasmid
DNA at quoted prices below $300/mg in milligram quantities and $15/mg in gram
quantities (early 2007).

Conformations:-

Plasmid DNA may appear in one of five conformations, which (for a given size) run at
different speeds in a gel during electrophoresis. The conformations are listed below in
order of electrophoretic mobility (speed for a given applied voltage) from slowest to
fastest:

• "Nicked Open-Circular" DNA has one strand cut.

• "Relaxed Circular" DNA is fully intact with both strands uncut, but has been
enzymatically "relaxed" (supercoils removed). You can model this by letting a
twisted extension cord unwind and relax and then plugging it into itself.
 • "Linear" DNA has free ends, either because both strands have been cut, or
because the DNA was linear in vivo. You can model this with an electrical
extension cord that is not plugged into itself.
• "Supercoiled" (or "Covalently Closed-Circular") DNA is fully intact with both
strands uncut, and with a twist built in, resulting in a compact form. You can
model this by twisting an extension cord and then plugging it into itself.
• "Supercoiled Denatured" DNA is like supercoiled DNA, but has unpaired regions
that make it slightly less compact; this can result from excessive alkalinity during
plasmid preparation. You can model this by twisting a badly frayed extension
cord and then plugging it into itself.

The rate of migration for small linear fragments is directly proportional to the voltage
applied at low voltages. At higher voltages, larger fragments migrate at continually
increasing yet different rates. Therefore the resolution of a gel decreases with increased
voltage.

At a specified, low voltage, the migration rate of small linear DNA fragments is a
function of their length. Large linear fragments (over 20kb or so) migrate at a certain
fixed rate regardless of length. This is because the molecules 'reptate', with the bulk of the
molecule following the leading end through the gel matrix. Restriction digests are
frequently used to analyse purified plasmids. These enzymes specifically break the DNA
at certain short sequences. The resulting linear fragments form 'bands' after gel
electrophoresis. It is possible to purify certain fragments by cutting the bands out of the
gel and dissolving the gel to release the DNA fragments.

Because of its tight conformation, supercoiled DNA migrates faster through a gel than
linear or open-circular DNA.
Inserting a DNA Sample into a Plasmid:-

Plasmids are similar to viruses, but lack a protein coat and cannot move from cell to cell
in the same fashion as a virus.

Plasmid vectors are small circular molecules of double stranded DNA derived from
natural plasmids that occur in bacterial cells. A piece of DNA can be inserted into a
plasmid if both the circular plasmid and the source of DNA have recognition sites for the
same restriction endonuclease.

The plasmid and the foreign DNA are cut by this restriction endonuclease (EcoRI in this
example) producing intermediates with sticky and complementary ends. Those two
intermediates recombine by base-pairing and are linked by the action of DNA ligase. A
new plasmid containing the foreign DNA as an insert is obtained. A few mismatches
occur, producing an undesirable recombinant.
The new plasmid can be introduced into bacterial cells that can produce many copies of
the inserted DNA . This technique is called DNA cloning.
pBR322: description & restriction map

pBR322 was one of the first truly useful cloning vectors. Pieced together by Bolivar and
Rodriguez at UNAM in the 1970’s, it contained all the characteristics of a good cloning
plasmid-selective marker, scorable marker, origin, and high copy number. As shown by
the picture above, there is a BamHI site specific to the tetracycline resistance gene; there
are also SalI and SphI sites specific there, as well.

we cleave the plasmid at the BamHI site, and ligate in our insert, this will disrupt the
tetracycline resistance gene. This means that cells sucessfully transformed with the
plasmid+insert won’t grow on tetracycline plates. Cells with just plain plasmid will.

However, tetracycline is bacteriostatic; that is, it doesn’t kill the cell outright, it just
prevents further growth. Cycloserine, on the other hand, is bacteriocidal; it will outright
kill growing cells. If we plate transformed cells on a tetracycline medium, only
transformed cells with plain plasmid will grow; we then treat the plate with cycloserine,
and those cells will suicide on the cycloserine, while the cells we do want, the
plasmid+insert, stay dormant.

We then transfer what’s left on the plate onto a fresh medium, this time with ampicillin,
and only the cells with plasmid + insert will grow (because wild-type cells can’t grow on
ampicillin, and the plain plasmid cells are already dead.

The plasmid pBR322 is one of the most commonly used E.coli cloning vectors. pBR322
is 4361 bp in length and contains: (1) the replicon rep responsible for the
replication of plasmid (source - plasmid pMB1); (2) rop gene coding for the Rop
protein, which promotes conversion of the unstable RNA I - RNA II complex to a
stable complex and serves to decrease copy number (source - plasmid pMB1); (3)
bla gene, coding for beta-lactamase that confers resistance to ampicillin (source -
transposing Tn3); (4) tat gene, encoding tetracycline resistance protein (source -
plasmid pSC101).

The circular sequence is numbered such that 1 is the first T of the unique EcoRI site
GAATTC and the count increases first through the tat gene, the pMB1 material, and
finally through the Tn3 region. The map shows enzymes that cut pBR322 DNA once.
Enzymes produced by Ferments are shown in blue. The coordinates refer to the position
of first nucleotide in each recognition sequence.
The exact position of genetic elements is shown on the map (termination cordons
included). The bla gene nucleotides 4153-4085 (complementary strand) code for a signal
peptide. The indicated rep region is sufficient to promote replication. DNA replication
initiates at position 2533 (+/- 1) and proceeds in the direction indicated. Plasmids
carrying the pMB1 and ColE1 replicas are incompatible, but they are fully compatible
with those carrying the p15A replicon (pACYC177, pACYC184).

The circular sequence is numbered such that 1 is the first T of the unique EcoRI site
GAATTC and the count increases first through the tet gene, the pMB1 material, and
finally through the Tn3 region. The map shows enzymes that cut pBR322 DNA once.
Enzymes produced by Fermentas are shown in blue. The coordinates refer to the position
of first nucleotide in each recognition sequence.

The exact position of genetic elements is shown on the map (termination codons
included). The bla gene nucleotides 4153-4085 (complementary strand) code for a signal
peptide. The indicated rep region is sufficient to promote replication. DNA replication
initiates at position 2533 (+/- 1) and proceeds in the direction indicated. Plasmids
carrying the pMB1 and ColE1 replicons are incompatible, but they are fully compatible
with those carrying the p15A replicon (pACYC177, pACYC184).

pMB1-derived plasmids can be amplified using chloramphenicol.

Enzymes which cut pBR322 DNA once:

AatII 4284, AflIII 2473, BamHI 375, BoxI 712, Bpu10I 1580, BsaAI 2225, BseJI
1668, BsgI 1650, Bsp68I 972, Bst1107I 2244, Bsu15I 23, BveI 1063, CaiI 2884,
Eam1105I 3361, Eco31I 3433, Eco32I 185, Eco52I 939, Eco88I 1425, Eco130I
1369, EcoRI 4359, Esp3I 2122, HindIII 29, Kpn2I 1664, MlsI 1444, Mva1269I
1353, NdeI 2295, NheI 229, PaeI 562, PfoI 2117, PscI 2473, PstI 3607, PsyI
2217, PvuI 3733, PvuII 2064, SalI 651, SapI 2350, ScaI 3844, SgrAI 409, SspI
4168, TstI 389, VspI 3537, XagI 622, XapI 4359
 References

• TEXT BOOKS:-
 1. Biotechnology-expanding horizons: b.d.singh. Kalyani publishers . 2nd
edition
 2. Molecular biotechnology: s.b.parimrose. Panima publication. 2nd
edition
 3. Microbiology: klein, donald w.; prescott. Mcgraw-hill.
4. Plasmids: current research and future trends. Caister academic press.

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