By Idiatu Balogun

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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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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
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Type IA - prokaryotic enzyme
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Type IB – eukaryotic enzyme
DNA gyrase also referred to as DNA topoisomerase II was discovered by Martin
Gellert

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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

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

TOPO cloning kit is available for PCR cloning with either Taq DNA
polymerase or a proofreading enzyme. Some examples are
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TOPO TA Cloning Kit
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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
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General subcloning
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Long PCR fragments
In vitro transcription
Sequencing
Expression in E. Coli
Expression in mammalian cells
Entry into Gateway systems

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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

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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

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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

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TOPO cloning for E. Coli expression
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.

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The BL21 Star(DE3) E. coli strain carries the DE3 bacteriophage lambda lysogen. This λ DE3 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

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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

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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.