Vorlesung „Grundlagen der Genetik“
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Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Part IV Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Reminder (Part I, II, III) - DNA-structure, replication - Enzymes: polymerases, ligases, restriction-endonucleases - basic steps in DNA-cloning (digestion, ligation) - labeling of DNA (PCR, PNK, Nick-translation, random-priming) - hybridization-techniques (FISH, SKY, Southern-blot, Northern-blot) - sequencing, genetic code, DNA to protein - transformation techniques (chemical, electroporation, biolistic) Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Vehicles for DNA: plasmids/vectors Vector: DNA-construct, enabling the transfer and propagation of „Target-DNA“ in host-cells (e.g. E. coli, yeast) often deduced from bacterial plasmids most are circular closed molecules need host-specific selectable markers (ampR, URA etc.) posses own origin of replication (ori, ars), specific for host system „Shuttle-vectors“ are suitable for different host organisms are specialized for particular applications Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Vehicles for DNA: plasmids/vectors LB-amp. plate containing X-Gal. Alpha-Komplementierung in E. coli Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Alpha-complementation: blue-white screening in E. coli ß-Galactosidase active, blue colonies ß-galactosidase with DM15 mutation, inactive, no blue colonies a-peptide of ß-galactosidase, inactive, no blue colonies ß-Galactosidase with DM15 mutation and a-peptide, active, blue colonies Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Yeast artificial chromosomes (YACs) for large (> 100 kb) DNA-fragments Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Expression vectors Allow the expression of a target DNA in a specific host Use of specific promotors (drives transcription of 3‘adjacent DNA-sequences, „switch on“) Often exploit inducible promotors (switch) Promotor Target DNA/gene, e.g. GFP Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann pET-expression system for E. coli Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Expression of fluorescent proteins via the pET-system in E. coli Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Expression vector for S. pombe In yeast often strains with appropriate genetic markers, i.e. with respective auxotrophic markers, are used. The plasmid (LEU2) complements the auxotrophic genomic mutation (leu2). Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Expression of EGFP-fusion genes in S. pombe for localization studies GFP Sp rad22+ GFP Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Expression vectors for S. cerevisiae - 2µ-plasmids with high-copy number Cen-ars plasmids as single copy number - Different promotors Mumberg et al., 1995, Gene 156: 199-122 Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Expression vectors for S. cerevisiae - Combination of copy number and promotor ⇒ different expression levels Promotor ß-Galactosidase Gene expression Determination enzyme activity Mumberg et al., 1995, Gene 156: 199-122 Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Appropriate promotor/marker gene combinations: whole cell sensors of S. cerevisiae - Driving the transcriptional activity dependent on specific signals - Cells sense a number off parameters (temperature, stress, availability of nutritions, etc.) - Often change of expression of specific genes (transporters, repair genes, etc.) - Such genes posses specific promotors/switches - e.g. availabilty of carbon sourcee Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann S. cerevisiae whole-cell sensor for glucose Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann S. cerevisiae whole-cell sensor for glucose Cells are monitoring the availability of glucose on the basis of EGFP under control of the HXK1-promotor Thierfelder et al., Appl Biochem Biotechnol. 2011 Apr;163(8):954-64 Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann S. cerevisiae pheromone based directed cell-communication - S. cerevisiae cells are of mating-type a or a - communication via pheromones and specific receptors - FIG1-promotor is acticated by pheromones Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann S. cerevisiae pheromone based directed cell-communication FIG1-Prom. without pheromone EGFP pheromone induction Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann S. cerevisiae pheromone based directed cell-communication and signal-amplification Groß et al ., 2011, Lett . Appl. Microbiol. 2011 May 52(5):521-526 Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Protein-based structures have a high potential for technical applications - in many cases hard to isolate - often only limited amounts available Challenge Obtain a special protein for technical apllications - in high amounts - as a pure preparation - with tailored modifications Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Solution - DNA-based expression - use of cell-factories - use of well-established purification methods Prerequisite Genetic information (DNA-sequence) of the protein of interest has to be known. Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Strategy - isolation of protein from natural host, protein sequencing - design of degenerated (deg.) primers - isolation of genomic DNA, PCR with deg. primers - identify a DNA-fragment encoding (part of) the protein - analyse genomic architecture (Southern-blot) - construct DNA-library - colony hybridization - identify positive clones - DNA sequence analysis, data interpretation Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann As an example: he story of sslA - - Surface-layer protein of Sporosarcina ureae - self-assembles to highly ordered protein films - exhibits metal-binding capacity - potential in nanotechnology protein expressed in yeast leads to wire-like structures Identification of sslA as an example! Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Getting information about SslA Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann The genetic code Each amino acid is coded by at least one codon (3 bases) on the RNA or DNA level Uracil in RNA corresponds to thymine in DNA Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Design of degenerated primers This set of primers covers all possible codes for a specific amino acid sequence Protein: Coding: Trp Tyr Gly Lys Trp Trp TGG TAT GGA AAA TGG TGG C G G C T Part of the set of degenerated primers: P1: P3: P5: P7: P9: TGG TGG TGG TGG TGG TAT TAT TAT TAT TAT GGA GGG GGC GGT GGT AAA AAA AAA AAA AAG TGG TGG TGG TGG TGG TGG TGG TGG TGG TGG P2: P4: P6: P8: P10: TGG TGG TGG TGG TGG TAC TAC TAC TAC TAC GGA GGG GGC GGT GGT AAA AAA AAA AAA AAG TGG TGG TGG TGG TGG TGG TGG TGG TGG TGG Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Identification of a (partial) gene fragment Isolation of genomic DNA of S. ureae as a template for PCR PCR-reactions with the primer set IfG, P. Ryzhkov, 2007 Isolation of specific fragments Sequence analysis of fragments Identification of a gene fragment hybridization probe Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Analysis of genomic architecture of the gene Isolation of genomic DNA Southern blot with fragment as a probe IfG, P. Ryzhkov, 2007 Size of hybridizing fragment is known! Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann Screening of a DNA-library! Isolated fragment serves as a hybridization probe for screening! Construction of DNA-libraries A mixture of DNA-fragments covering the genetic information of an organism is cloned into a vector.
