The practical proceeded in five sessions. The first session involved DNA manipulation, which involved two steps. The aim of the first step, small-scale plasmid isolation (â€˜Miniprepâ€™), was to isolate a recipient plasmid, pBlue Script KS II (+), from anÂ E. coliÂ culture prepared the previous day. In the second exercise, a double restriction endonuclease digestion of the two plasmids, i.e., the donor (pProEX) and the recipient (pBKSII), was done usingÂ BamHI andÂ HindIII. The aims of this step were to excise the gene of interest (Pc-FtsZ) from pProEX and prepare the recipient plasmid, pBKSII, for cloning. The two restriction endonucleases cleaved the DNA at specific sites to generate sticky ends that allowed the foreign fragment (Pc-FtsZ) to be inserted into the recipient plasmid.
Practical two involved the construction of a recombinant vector, pBKS II-ftsZ,Â using the restriction digests from the first lab session. Its aims were to compare the relative molecular sizes of the restriction digests with standards, confirm whether the restriction digestion was successful, and separate and purify the gene of interest for ligation. It entailed agarose gel electrophoresis to resolve the DNA into separate bands, excision to remove the desired fragments, and ligation of the gene into the pBKSII plasmid. The new pBKSII plasmid contained theÂ Pc-ftsZÂ gene and could be inserted intoÂ E. coliÂ in the next session.
The recombinant plasmids (containingÂ Pc-ftsZ gene)Â from the above step were used to transformÂ E. coliÂ cells made competent through heat shock. The practical proceeded in two steps: (1) bacterial transformation (cloning) and (2) Southern blotting analysis of the cloned DNA. The objectives were to introduce ligated vector (pBKSII) intoÂ E. coliÂ for cloning and analyse the cloned inserts using agarose gel electrophoresis and Southern blotting.
Since not all cells in the last session could take up the ligated vector, identifying transformed cells from the non-transformed ones was important. In the fourth practical session, an analysis of the Southern blot was done using specific probes. The main objective of this step was to confirm the presence ofÂ PC-FtsZÂ in the transformed bacteria. The practical also involved the screening of transformed bacteria using PCR to identify those carrying the pBKS II-Pc-ftsZÂ construct. The final (fifth) practical involved the screening of the Southern blot (gel) and testing theÂ TaqÂ polymerase colonies to confirm the results obtained in the previous session. Running a gel of the PCR colony screen obtained in the previous practical helped identify specific sequences in the transformed bacteria.