Genetic engineering 2600-S1-CM-IGN

During the laboratory classes, the principles of good laboratory practice (GLP) and the technique of pipetting with automatic pipettes will be discussed at the beginning. During the classes, students will learn about methods of extracting nucleic acids from plant material and microorganisms, they will independently perform DNA amplification using PCR, restriction digestion, ligation, transformation. They will learn how it is possible to check the potential function of a cloned gene by performing a functional analysis in a prokaryotic system using Escherichia coli. Students will learn about the principles of working with genetically modified organisms. The following topics will be covered during the laboratory classes:

1. Introduction. Good laboratory practice. Pipetting technique with automatic micropipettes. Preparation of solutions.
2. Amplification of the rapeseed metallothionein 3 (MT3) coding region on a cDNA template (cloned into the pJET 1.2 vector) using primers generating restriction sites for the NdeI and XhoI enzymes. 3. Restriction digestion of the pET21 expression plasmid and the PCR reaction product (insert) and their ligation
4. Competence of E. coli cells and their transformation with the ligation mixture
5. Isolation of pET21 plasmid DNA by alkaline lysis and its restriction digestion
6. Electrophoresis of restriction digestion products and functional analysis of BL21 bacteria carrying the pET21 plasmid with the BnMT3 coding sequence insert on medium containing heavy metals
7. Functional analysis of Escherichia coli bacteria expressing BnMT3 (continued) and in silico analysis of the sequence containing the MT3 gene of Brassica napus L.