10.026.13-16 Regulierbare Promotoren zur Kontrolle der Genexpression in Hefe (Hefepraktikum)

Lehrende/r: Univ.-Prof. Dr. Petra Beli; Univ.-Prof. Dr. Brian Luke; Dr. Natalie Schindler

Veranstaltungsart: Vorlesung/Seminar/Übung

Anzeige im Stundenplan: 10.026.13-16

Semesterwochenstunden: 11

Credits: 15,0

Unterrichtssprache: Englisch

Min. | Max. Teilnehmerzahl: - | 12

Voraussetzungen / Organisatorisches:
Blockveranstaltung, 2 Wochen, 9:15-17:00 Uhr, Mo.-Fr., 26.08.-06.09.2024, Biozentrum 1, Hanns-Dieter-Hüsch-Weg 15, EG, Lehrlabor 1 (Raum 00.385) und SR 2 (Raum 00-307)
Vorbesprechung und Themenvergabe für Seminar: Mo., 29.07.2024, 11.00-12.00 Uhr, digital in MS-Teams, weitere Informationen im LMS-Moodle zur Veranstaltung

Inhalt:
The participants will get insight into the applied molecular genetics in budding yeast and will genetically modify different genes of interest using natural homologous recombination. The aim is to modify genes with a Galactose inducible promoter and an epitope tag that will allow steering gene expression and facilitates the detection of the gene products. The students will work out all steps of the process to generate such yeast strains during the course guided by lectures that provide the required theoretical information. The students will design the cloning strategy of the knock-in cassette, perform the PCR reaction, cultivate yeast, transform yeast, analyze the transformants via colony PCR and finally will test gene expression and the functionality of the novel strains by SDS-PAGE and Western blot analyses. To gain a solid knowledge on the general context of their topic, the students will present a scientific paper about their gene of interest to the group. The course will transfer good laboratory practice skills with focus on how to keep a lab journal.
Content:

• In silico planning of PCR approaches to generate knock-in cassettes
  
• Execution of PCR and agarose gel electrophoresis
  
• Transformation of competent yeast and selection of transformants using auxotrophy
  
• Confirmation of the genetic modification by yeast colony PCR
  
• Checking transcriptional regulation by selective induction of gene expression, and SDS-PAGE and Western blot as key tool for protein separation and analysis using antibody detection
  
• Documentation of experimental workflow, data interpretation and final report of results and their discussion in the light of the current state of the art research