Transcript TOPO CLONING - POKEWEED-ANTIVIRAL

TOPO CLONING
By Idiatu Balogun
INTRODUCTION
The structure of DNA is a double-stranded
helix, where in the four bases, adenine, thymine,
guanine, and cytosine are paired and stored in
the centre of this helix. While this structure
provides a stable means of storing the genetic
code, Watson and Crick noted that the two
strands of DNA are intertwined, and this would
require the two strands to be untwisted in
order to access the information stored
 In biological systems reaction occurs which are
catalysed by enzymes known as DNA
topoisomerases.
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BRIEF HISTORY
The first DNA topoisomerase 1was discovered
by James Wang in 1971, the ω protein from
Escherichia coli
 ‘nicking-closing’ activity from nuclear extracts of
mouse cells by James Champoux also referred to
as DNA topoisomerase I
These two enzymes are now classified in different
 Type IA - prokaryotic enzyme
 Type IB – eukaryotic enzyme
 DNA gyrase also referred to as DNA
topoisomerase II was discovered by Martin
Gellert
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TOPOISOMERASE
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A class of enzymes that alter the supercoiling of doublestranded DNA. (In supercoiling the DNA molecule coils up like a
telephone cord, which shortens the molecule.) The topoisomerases
act by transiently cutting one or both strands of the DNA.
Topoisomerase type I cuts one strand whereas topoisomerase type
II cuts both strands of the DNA to relax the coil and extend the
DNA molecule.
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The regulation of DNA supercoiling is essential to DNA
transcription and replication, when the DNA helix must unwind to
permit the proper function of the enzymatic machinery involved in
these processes. Topoisomerases serve to maintain both the
transcription and replication of DNA.
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Aside from topoisomerases I and II, there are more discovered
topoisomerases. Topoisomerase III may regulate recombination
while topoisomerase IV regulates the process of segregating newly
replicated chromosomes from one another
DEFINITION
TOPO cloning is the enzyme DNA topoisomerase I, which
functions both as a restriction enzyme and as a ligase. Its biological
role is to cleave and rejoin DNA during replication. Vaccinia virus
topoisomerase I specifically recognizes the pentameric sequence
5´-(C/T)CCTT-3´ and forms a covalent bond with the phosphate
group attached to the 3´ thymidine. It cleaves one DNA strand,
enabling the DNA to unwind. The enzyme then religates the ends
of the cleaved strand and releases itself from the DNA. To harness
the religating activity of topoisomerase, TOPO vectors are
provided linearized with topoisomerase I covalently bound to each
3´ phosphate. This enables the vectors to readily ligate DNA
sequences with compatible ends The ligation is complete in only 5
minutes at room temperature
TOPO TA cloning of Taq-amplified DNA
Zero Blunt TOPO cloning of blunt-end DNA
Directional TOPO cloning of blunt-end DNA
3 Steps in TOPO Cloning
TOPO Cloning Kits
TOPO cloning kit is available for PCR cloning with either Taq DNA
polymerase or a proofreading enzyme. Some examples are
 TOPO TA Cloning Kit
 Zero Blunt TOPO PCR Cloning Kit
 Champion™ pET100 Series Directional TOPO Expression Kit
And many more can be found on the website
www.invitrogen.com/topo
Also selecting the right TOPO cloning kit depends on the application
of cloned PCR product for example
 General subcloning
 Long PCR fragments
 In vitro transcription
 Sequencing
 Expression in E. Coli
 Expression in mammalian cells
 Entry into Gateway systems
pET DIRECTIONAL TOPO
EXPRESSION KITS
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are used and depending on the vector chosen, the
pET TOPO vectors are available with:
N-terminal or C-terminal peptide tags for
production of recombinant
fusion proteins that may be easily detected or
purified
Protease recognition site for removal of the Nterminal peptide tag from
recombinant fusion protein
Antibiotic resistance marker for selection of
transformants
Some examples of pET TOPO vector used
are:
 pET100/D-TOPO
 pET200/D-TOPO
 pET101/D-TOPO
 pET102/D-TOPO and
 pET151/D-TOPO which we will be and
allows expression of recombinant protein
with an N-terminal tag containing the V5
epitope and a 6xHis tag. The N-terminal tag
also includes a TEV protease cleavage site to
enable removal of the tag after protein
purification using TEV protease
Map and Features of pET151/DTOPO
HOW DIRECTIONAL TOPO
CLONING WORKS
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PCR products are directionally cloned by
addition of 5’-CACC to the upstream
primer hence the downstream primer
does not contain this terminal sequence
and the PCR product is incubated with
TOPO- activated pET vector in which
one end contains a 3’ overhang GTGG
whereby annealing to CACC whilst the
topoisomerase catalyses the formation of
phosphodiester link
TOPO cloning for E. Coli
expression
T7-Regulated Expression
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In the pET TOPO vectors, expression of the gene
of interest is controlled by a strong bacteriophage
T7 promoter that has been modified to contain a
lac operator sequence. To express the gene of
interest, it is necessary to deliver T7 RNA
polymerase to the cells by inducing expression of
the polymerase or infecting the cell with phage
expressing the polymerase. T7 RNA polymerase is
supplied by the BL21 Star(DE3) host E. coli strain
in a regulated manner . When T7 RNA
polymerase is produced, it binds to the T7
promoter and transcribes the gene of interest.
Regulating expression of T7 RNA
Polymerase
The BL21 Star(DE3) E. coli strain carries the DE3 bacteriophage
lambda lysogen. This λDE3 lysogen contains
 a lac construct consisting of the following elements:
 the lacI gene encoding the lac repressor
 the T7 RNA polymerase gene under control of the lacUV5
promoter
 a small portion of the lacZ gene.
This lac construct is inserted into the int gene such that it inactivates
the int gene. Disruption of the int gene prevents excision of the
phage (i.e. lysis) in the absence of helper phage. The lac repressor
(encoded by lacI) represses expression of T7 RNA polymerase.
Addition of the gratuitous inducer, isopropyl β-D-thiogalactoside
(IPTG) allows expression of T7 RNA polymerase from the lacUV5
promoter. The BL21 Star(DE3) strain also contains other features
which facilitate high-level expression of heterologous genes
T7lac Promotor and Expressing
toxic genes
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The presence of T7 RNA polymerase at
basal levels can lead to expression of the
desired gene even in the absence of inducer.
If the gene of interest is toxic to the E. coli
host, plasmid instability and/or cell death
may result.
Using TOP10 Cells competent E. coli which
do not contain T7 RNA polymerase are
included in each pET Directional TOPOR
Expression kit to provide a host for stable
propagation and maintenance of
recombinant plasmids
The BL21 Star (DE3) E. coli strain is included
in pET Directional TOPO Expression Kit for
use as a host for expression
 Gene toxic to BL21 Star (DE3) cells are
expressed in the BL21 Star(DE3)pLysS, which
contains the pLysS plasmid producing T7
lysozyme. T7 lysozyme binds to T7 RNA
polymerase and inhibits transcription. This
activity results in reduced basal levels of T7
RNA polymerase, leading to reduced basal
expression of T7-driven heterologous genes.
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