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Fundamental of Biotechnology
Plasmids
Introduction
 The
term plasmid was
first introduced by the
American molecular
biologist Joshua
Lederberg in 1952.
Introduction
 Plasmid
 Self
replicating, double
Circular DNA molecule
 extrachromosomal
(bacteria, yeast)
stranded,
entities in the cell
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 Size
from less than 1kb to more than
500kb. (approx 0.1to 5% of the total DNA)

Like the host-cell chromosomal DNA, plasmid
DNA is duplicated before every cell division.

at least one copy of the plasmid DNA is
segregated to each daughter cell
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#
of identical plasmids within a single
cell can range from one to even
thousands
(under some circumstances).

Called high copy number plasmid (10-100 copies per host cell)
Low copy number plasmid (1-4 copies per cell)

Carry information for their own transfer (F Plasmid)

Resistance to antibiotics (R Plasmid)

Narrow and Broad Host Range Plasmids

Due to the specificity of origin of replication:

Narrow host range Plasmid: Replicate in only
one spices of host cell.

Broad host range Plasmid: less specific origin
of replication
Conjugative / Non conjugative

Conjugative: self transmissible, tra (transfer)
and mob (mobilising) regions carried on the
plasmid.

Non conjugative: not self transmissible by
conjugative proficient plasmid if their mob
region is functional.
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 Conjugative
plasmid large= Low copy
number
 Non conjugative small= high copy
number
Plasmid Cloning Vectors

Plasmid cloning vectors have to be genetically
engineered

Naturally occurring plasmids often lacks important
features such as

Small size (>15kb decrease transfer in E. Coli)
Choice of unique restriction site (to clone the DNA of
interest)


One or more selectable markers (to identify the
recipient cells)
MCS
Plasmids cloning vector
ORI
Ab. Resis
Three features of the plasmid cloning vectors:

Multiple cloning site. The place where foreign DNA
fragments can be inserted.

An origin of replication: The replication origin is a
specific DNA sequence of 50-100 base pairs that must be
present in a plasmid for it to replicate. Host-cell enzymes
bind to ORI, initiating replication of the circular DNA.

A gene specifying resistance to an Antibiotic. This
permits selective growth of the host cell.
Most often used: Resistance to ampicillin, penicillin,
tetracycline, kanamycin, and chloramphenicol.
A plasmid vector for cloning
1. Contains an origin of replication, allowing
for replication independent of host’s
genome.
2. Contains Selective markers: Selection of
cells containing a plasmid
Contains a multiple cloning site (MCS)
3. Easy to be isolated from the host cell.
Plasmid vectors
Nomenclature
Plasmid is designated by lower case such as
p (stands for plasmid).
Descriptive abbreviation some time e.g.
pBR322
BR recognises the research work of F. Boliver
and R. Rodriguez.
322 relevance to these workers.
Plasmid vector pBR322
First plasmid vectors to be developed was
pBR322 (Bolivar et al., 1977),
constructed by ligating restriction fragments
from three naturally occurring E. coli plasmids:
R1, R6.5 and pMB1.
The pBR322 plasmid is small (just 4361 bp)
having the origin of replication, markers for
double selection such as two antibiotics:
Ampicillin and Tetracycline
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


pBR322
Restriction sites
Recognition sequences for
seven restriction enzymes,

six of these sites lie within one or
other of the genes for antibiotic
resistance.

Tetr (BamHI, HinIII and SalI),
Ampr (Pst I, PvuI, Sca I)
Ecor1 in non coding region.

Cloning DNA Into a
Plasmid Vector

To reduce unwanted
product, treatment with
alkaline phosphate to
remove 5 prim phosphate
group from linear
plasmid DNA
pAT 153

Deletion derivatives of pBR322.

Removal of two fragments (705bp) by
using HaeII.

3 fold increase in copy number.

Better than pBR322.
Other plasmid cloning vectors
pBR322 was a well conceived cloning vector but has few
cloning sites
selection procedure is time consuming
pUC19 is a plasmid cloning vector
created by Messing and co-workers in the University of
California.
p =for plasmid and UC represents the University of
California.
circular double stranded DNA and has 2,686 base pairs
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pUC19 : recombinants, can be easily distinguished from the nonrecombinants based on colour differences of colonies on growth
media.
pUC18 is similar to pUC19, but possess different restriction sites.
Component of pUC 19

It has one ampR gene (ampicillin resistance gene),
lac Z gene of E. coli and lac I gene.

The multiple cloning site (MCS) or (polylinker) region
is split into the lac Z gene (codons 6-7 of lac Z are
replaced by MCS)

The multiple cloning site (e.g. EcorI, SacI, KpnI,
XmaI, SmaI, BamhI, XbaI, SalI, HincII, AccI, BspMI,
PstI, SphI, HindII)

origin of replication from pBR322.
Multiple Cloning Sites
Transformation and Selection
Transformed cell will grow b.c ampicillin resistance (ampR)
gene.
Transformed of interest can be distinguished by looking at
the colour of the colony they make on agar media.
Recombinants will be white, whereas non-recombinants will
bed blue in colour.
This is the most notable feature of pUC19.
Mechanism
In the presence of Isopropyl β-D-1-thiogalactopyranoside (IPTG)
in growth medium, bacteria synthesise both fragments of the
enzyme.
Both the fragments can together hydrolyse X-gal (5-bromo-4-chloro3-indolyl- beta-D-galactopyranoside) and form blue colonies on
media with X-gal.
Insertion of foreign DNA into the MCS located within the lac Z gene
causes insertional inactivation of this gene at the N-terminal
fragment of beta-galactosidase.
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Thus bacteria carrying recombinant plasmids in
the MCS cannot hydrolyse X-gal,
giving rise to white colonies, which can be
distinguished on culture media from nonrecombinant cells, which are blue.
Therefore the media used should contain ampicillin,
IPTG, and X-gal.
Antibiotics Commonly Used as Selective Agents
Plasmid vectors advantages and
disadvantages

Advantages:
• Small, easy to handle
• Straightforward selection strategies
• Useful for cloning small DNA fragments
(< 10kbp)

Disadvantages:
• Less useful for cloning large DNA fragments
(> 10kbp)