LESSONS (3 ECTS):
RECOMBINANT PROTEINS IN PROCARIOTS AND THEIR BIOTECHNOLOGICAL APPLICATIONS
-study of gene function and production of recombinant proteins
-procaryotic expression systems: Manipulation of gene expression in prokaryotes.
- Characteristics of prokaryotic expression vectors: strong and adjustable promoters (tac promoter, lac and pET binary system). Fusion proteins. Improved expression levels of recombinant proteins.
EUCARIOTIC EXPRESSION SYSTEMS AND BIOTECHNOLOGICAL APPLICATIONS
- Characteristics of vectors for expression in eukaryotes. Techniques for gene transfer in eukaryotes: chemical methods (transfection with calcium chloride or using liposomes); physical methods (electroporation, microinjection, genegun)
- Yeast (S. cerevisiae and P. pastoris): Vectors for expression in yeast. Selection markers (complementation of an auxotrophy).
- Insect cells: AcMNPV virus (Baculovirus) and SfN9 cells.
- Mammalian cells. Plasmid and viral vectors. Promoters for expression in eukaryotes. The Tet-ON / Tet-OFF system and promoters responsive to steroid hormones (tamoxifen).
- Transgenic animals: Homologous and site-specific recombination
Transgenic mice: How a transgenic mouse is produced, Inducible transgenic mice
Models of mice with an altered gene: knockout mice, knockin mice, conditional knockdown mice.
Other applications of transgenic animal technology: Transgenic primates, transgenic cattle, Pharming gene
- Cloning by nuclear transfer and - Applications Transgenic plants: Transfer of genes mediated by T-DNA. Electroporation and microbalistics
RNA REGULATORS IN PROCARIOTES AND EUCARIOTS
- The RNA-mediated regulation in prokaryotes: riboswitches; CRISPR-cas. System
Regulation mediated by RNA in eukaryotes:
- Mechanisms of silencing of a gene mediated by RNAi.
- Biotechnological applications of regulatory RNA: the revolution of the CRISPR / Cas9 system and genome editing; In vivo siRNA vehiculation.
LABORATORY (2 ECTS): Cloning of the sequence coding for SOD1 in the prokaryotic expression vector pET41b. Design of oligonucleotides for PCR cloning. PCR from plasmid. Digestion with restriction enzymes. Ligase. Bacterial transformation. Plasmid DNA extraction and analysis by restriction enzymes. Expression of recombinant proteins in E.coli. Analysis by SDS / PAGE and staining with Coomassie Blue. Activity gel for SOD1.
EXERCISE (1 ECTS): definition of Bioinformatics; databases; identification of sequences, design specific primers. Bioinformatics as a tool for analyzing sequences. NCBI databases (GeneBank, Unigene, PubMed, BLAST etc etc) and ENSEMBL. The SNAPGENE program for the restriction analysis of a DNA fragment. Cloning strategies by PCR