BISC209: Lab4
LAB 4: Con't. Project: Soil Microbial Communities & Diversity
Your instructor will return your frozen pcr products containing amplified fragments of 16s rDNA from many of the species of soil bacteria in your soil sample. Today you will insert your bacterial 16s rDNA fragments into a patented cloning vector (pCR-BluntII TOPO®) and then transform that vector into a special genetically engineered strain of Escherichia coli bacteria that will express a vector gene for kanamycin resistance, allowing us to select for transformants on media containing kanamycin.
The principle behind TOPO® cloning is the enzyme DNA topoisomerase I, which will function in this system 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 quickly ligate DNA sequences with compatible ends.
We used a polymerase that creates blunt ended DNA fragments rather than using TaQ, which makes fragments with 3' T overhangs. If you have single stranded T rich ends, complementary single stranded A rich "sticky ends" allow ligation. Blunt ends require a different Blunt-fragment cloning kit. Invitrogen's Zero Blunt® TOPO® PCR Cloning Kit should work well for us. It uses EcoRI restriction endonuclease recognition sequences flanking the pcr products target insertion site for easy removal of the insertion and it has several (T7, SP6, and M13 forward and reverse) priming sites for sequencing. It has two resistance genes, Kanamycin and Zeocin, for selction in E. coli.
Additionally, the cloning system we will use contains a background reducer, a lethal ccdB (control of cell death) encoding a ccdB protein that poisons bacterial DNA gyrase, causing degradation of the host chromosome and cell death. However, when one of our pcr products is ligated into the vector, the ccdB gene is disrupted, enabling these recombinant colonies to grow while other non-transformants do not. (Cool technology; yes!?)
Protocol for using Zero Blunt TOPO PCR Clonging Kit with One Shot TOP 10 Chemically Competent E. coli
PCR cloning requires three steps.
1. Add 1 μl of PCR reaction to 1 μl of TOPO® cloning vector.
2. Incubate 5 min at room temperature.
3. Transform the competent E. coli provided.
Protocol for Transforming TOPO Competent E. coli
1.
