E. coli plasmid based vectors and selection strategies for identification of correct recombinant

1. Plasmids
Plasmids are extrachromosomal, double stranded circular DNA molecules that carry
an origin of replication and replicate within bacterial cell (origin of replication is the
DNA sequence in a genome at which replication is initiated).
Properties of plasmids
a) Integration
 Some plasmids do not insert themselves into the bacterial chromosome,
they are called non-integrative plasmids.
 Integrative plasmids or episomes are able to replicate by inserting
themselves into bacterial chromosome.
b) Copy number
 Copy number is the number of molecules of an individual plasmid that
are normally found in a single bacterial cell. On the basis of copy
number plasmids can be divided into two types:
 Stringent plasmids: these have a low copy number, mostly one or two
per cell.
Relaxed plasmids: these are present in multiple copies of 50 or more
per cell.
c) Conjugation
 Conjugative plasmids promote conjugation between bacterial cells that
can help the plasmid to spread from one cell to all the other cells in a
bacterial culture. (Conjugation and plasmid transfer are controlled by a
set of transfer or tra genes).
 Non-conjugative plasmids lack tra genes and can not promote
conjugation.
d) Compatibility
 Different kinds of plasmids may be present in a single cell. For
co-existence in the same cell, different plasmids must be compatible.
 If two plasmids are incompatible then one or the other will be lost from
the cell.
Types of plasmids based on main characteristics coded:
 Fertility or F plasmids
 Resistance or R plasmids
 Colicin or Col plasmids
 Degradative plasmids
 Virulence plasmids
The best characterized eukaryotic plasmid that is found in yeast Saccharoyces
cerevisiae is 2µm circle.

2. Cloning Vectors Based on E. coli Plasmids
A) pBR series
1) pBR322
p= plasmid, BR= Boliver and Rodriguez, the two researchers who developed
pBR322,
322= the number distinguishes the plasmid vector from others developed in
same laboratory.
Figure: A map of pBR322
 pBR322 contains DNA derived from three different naturally occurring
plasmids:
ampR gene – from plasmid R1,
tetR gene – from plasmid R6-5
Origin of replication – from pMB1 (closely related to the colicin-producing
plasmid ColE1)
 It is a conjugative plasmid.
Advantages of pBR322:
a) Small size: pBR322 is 4363 bp, which means that the vector along with
recombinant DNA molecules can be purified with ease. Even with 6 kb of
additional DNA, a recombinant pBR322 molecule is still a manageable size.
b) Selectable markers: it carries two sets of antibiotic resistance genes. Either
ampicillin or tetracycline resistance can be used as a selectable marker for
cells containing the plasmid.
c) Multiple restriction sites:
Each marker gene includes unique restriction sites.

3.  Insertion of new DNA into pBR322 that has been restricted with PstI, PvuI, or
ScaI inactivates the ampR gen.
 Iinsertion using any one of eight restriction endonucleases (notably BamHI
and HindIII) inactivates tetracycline resistance.
 The great variety of restriction sites that can be used for insertional
inactivation means that pBR322 can be used to clone DNA fragments with any
of several kinds of sticky end.
d) High copy number: pBR322 has a reasonably high copy number. Generally
there are about 15 molecules present in a transformed E. coli cell, but this
number can be increased, up to 1000–3000, by plasmid amplification in the
presence of a protein synthesis inhibitor such as chloramphenicol.
2) pBR327
 pBR327 was produced by removing a 1089 bp segment (from bp 1427 to 2516)
from pBR322. It has a size of 3274 bp.
 This deletion left the ampR and tetR genes intact, but changed the replicative
and conjugative abilities of the resulting plasmid.
As a result, pBR327 differs from pBR322 in two important ways:
1) pBR327 has a higher copy number than pBR322, about 30–45 molecules
per E. coli cell. The higher copy number of pBR327 in normal cells makes
this vector more suitable if the aim of the experiment is to study the
function of the cloned gene because the more copies there are of a cloned
gene, the more likely it is that the effect of the cloned gene on the host cell
will be detectable.
2) The deletion also makes pBR327 a non-conjugative plasmid. This is
important for biological containment, preventing the risk of a
recombinant pBR327 molecule escaping from the test tube and colonizing
bacteria in the gut of a careless molecular biologist. pBR327 is
preferable if the cloned gene is potentially harmful should an accident
occur.
There are many more vectors in this series.

4. Selection strategy for pBR recombinants:
 Either ampR or tetR can be used for selection of pBR recombinants by
insertional inactivation of either gene.
 BamHI cuts within the cluster of genes that code for resistance to tetracycline.
A recombinant pBR322 molecule, that carries DNA insert in the BamHI site,
can no longer provide tetracycline resistance to its host, as one of the
necessary genes is now disrupted by the inserted DNA.
 Cells containing this recombinant pBR322 molecule are still resistant to
ampicillin, but sensitive to tetracycline (ampRtetS ).

5. Figure: Selection of pBR recombinants
 After transformation the cells are plated onto ampicillin medium and
incubated until colonies appear. All of these colonies are transformants
(untransformed cells are ampS and so do not produce colonies on the selective
medium).
 To identify the recombinants the colonies are replica plated onto tetracycline
containing agar medium. After incubation, some of the original colonies
regrow, but others do not. The colonies that do not grow on tetracycline agar
are recombinants (ampR
tetS
); once their positions are known, samples for
further study can be recovered from the original ampicillin agar plate.

6. B) pUC vectors
p= plasmid, UC= University of California
pUC8
 pUC8 is descended from pBR322, it contains the replication origin and the
ampR gene of pBR322.
 The ampR gene of pUC8 does not contain the unique restriction sites.
 All the cloning sites are clustered into a short segment of the lacZ′ gene
carried by pUC8.
Figure: pUC vector
Advantages of pUC8
a) High copy number - pUC8 has a high copy number of 500–700 resulting in
high yield of cloned DNA from E. coli cells transformed with recombinant
pUC8 plasmids.
b) Single step recombinant selection- Identification of recombinant cells can be
achieved by a single step process, by plating onto agar medium containing
ampicillin plus X-gal. A cloning experiment with pUC8 can therefore be
carried out in half the time needed with pBR322 or pBR327.
c) DNA with different sticky ends can be cloned- The restriction sites are
clustered, which allows a DNA fragment with two different sticky ends (for
example EcoRI at one end and BamHI at the other) to be cloned without using
additional manipulations such as linker attachment.
d) Single stranded form of Gene can be obtained by M13mp counterpart
-The restriction site clusters in these vectors are the same as the clusters in the
equivalent M13mp series of vectors. DNA cloned into a member of the pUC

7. series can therefore be transferred directly to its M13mp counterpart, enabling
the cloned gene to be obtained as single-stranded DNA.
Selection for pUC recombinants (Lac selection or
α-complementation)
 pUC8 carries the ampicillin resistance gene and a gene called lacZ′, which
codes for part (α- subunit) of the enzyme β-galactosidase.
 Cloning with pUC8 involves insertional inactivation of the lacZ′ gene.
Recombinants are identified because of their inability to synthesize
β-galactosidase.
 β-Galactosidase is involved in the breakdown of lactose to glucose and
galactose. It is normally coded by the gene lacZ, which resides on the E. coli
chromosome.
 Mutant host E. coli have a modified lacZ gene,that lacks the lacZ′ segment of
β- galactosidase. These mutants can synthesize the enzyme only when they
harbor a plasmid, such as pUC8, that carries the missing lacZ′ segment of the
gene.
 Screening for β-galactosidase presence involves a lactose analog called X-gal
(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) which is broken down
by β-galactosidase to a product that is colored deep blue.

8. Figure: Lac Selection of pUC recombinants
Figure: Lac Selection
 After transformation the cells are plated onto media containing X-gal, IPTG
(isopropylthiogalactoside, an inducer of the enzyme) and ampicillin.

9.  The non-recombinant colonies (the cells of which synthesize β-galactosidase),
will be colored blue, whereas recombinants (with a disrupted lacZ′ gene and
unable to make β-galactosidase), will be white.
 This system is called Lac selection. Both ampicillin resistance and the
presence /absence of β-galactosidase are tested for on a single agar plate.
C) pGEM3Z vectors
pGEM3Z is very similar to a pUC vector. It carries the ampR and lacZ′ genes.
It is2750 bp in size. lacZ′ region contains a cluster of restriction sites.
Figure: a map of pGEM3Z vector (R = cluster of restriction sites for EcoRI, SacI, KpnI, AvaI,
SmaI, BamHI, XbaI, SalI, AccI, HincII, PstI, SphI, and HindIII).
 pGEM3Z has two promoter sequences that lie on either side of the cluster of
restriction sites. Each promoter acts as the recognition site for attachment of
an RNA polymerase enzyme.
Fig: in vitro RNA synthesis
 pGEM3Z and other vectors of this type carry one promoters that is specific for
the RNA polymerase coded by T7 bacteriophage and the other for the RNA
polymerase of SP6 phage. These RNA polymerases are synthesized during
infection of E. coli with one or other of the phages.

10.  They are chosen for use in in vitro transcription as they are very active
enzymes. These RNA polymerases are able to synthesize 1–2 mg of RNA per
minute, substantially more than can be produced by the standard E. coli
enzyme.
Advantages of pGEM3Z
 These vectors can be used for production of RNA transcripts of desired gene.
If a recombinant pGEM3Z molecule is mixed with purified RNA polymerase
in the test tube, transcription occurs and RNA copies of the cloned fragment
are synthesized.
 The RNA that is produced could be used as a hybridization probe, or might be
required for experiments aimed at studying RNA processing (e.g., the removal
of introns) or protein synthesis.
Selection strategy for pGEM3Z: same as for pUC vectors (Lac selection).
REFERENCE:
Gene Cloning And DNA Analysis An Introduction, T.A. Brown , 6th
edition