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  1. TOPO CLONING By IdiatuBalogun

  2. 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.

  3. BRIEF HISTORY • The first DNA topoisomerase1was 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 topoisomeraseI These two enzymes are now classified in different • Type IA -prokaryotic enzyme • Type IB – eukaryotic enzyme • DNA gyrasealso referred to as DNA topoisomerase II was discovered by Martin Gellert

  4. TOPOISOMERASE • A class of enzymes that alter the supercoiling of double-stranded 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. • 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. • 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

  5. DEFINITION TOPO cloning is the enzyme DNA topoisomeraseI, which functions both as a restriction enzyme and as a ligase. Its biological role is to cleave and rejoin DNA during replication. Vacciniavirus 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 ligateDNA sequences with compatible ends The ligation is complete in only 5 minutes at room temperature

  6. TOPO TA cloning of Taq-amplified DNA Zero Blunt TOPO cloning of blunt-end DNA Directional TOPO cloning of blunt-end DNA

  7. 3 Steps in TOPO Cloning

  8. 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 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

  9. pET DIRECTIONAL TOPO EXPRESSION KITS • are used and depending on the vector chosen, the pETTOPO 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 N-terminal peptide tag from • recombinant fusion protein • Antibiotic resistance marker for selection of transformants

  10. Some examples of pETTOPO 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

  11. Map and Features of pET151/D-TOPO

  12. HOW DIRECTIONAL TOPO CLONING WORKS • 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

  13. TOPO cloning for E. Coli expression T7-Regulated Expression • In the pET TOPO vectors, expression of the gene of interest is controlled by a strong bacteriophageT7 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.

  14. Regulating expression of T7 RNA Polymerase The BL21 Star(DE3) E. coli strain carries the DE3 bacteriophage lambda lysogen. This λDE3 lysogencontains • 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 intgene. 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

  15. T7lac Promotor and Expressing toxic genes • 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

  16. The BL21 Star (DE3) E. coli strain is included in pETDirectional 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 heterologousgenes.