| Ansvarligt institut | Institut for Plantebiologi og Bioteknologi
80 % Institut for Basal Husdyr- og Veterinærvidenskab 12 % Institut for Fødevarevidenskab 8 % | ||||||||||||||||
| English Title | Heterologous Expression | ||||||||||||||||
| Tidligst mulig placering | Kandidat 1.år til Kandidat 2. år | ||||||||||||||||
| Varighed | En blok | ||||||||||||||||
| Pointværdi | 15 (ECTS) | ||||||||||||||||
| Kursustype | Kandidatkursus | ||||||||||||||||
| Eksamen | Sluteksamen mundtlig eksamen Alle hjælpemidler tilladt Beskrivelse af eksamen: The student will be examined in one of the practical exercises as well as one of the theoretical cases. Following there will be a discussion where the selected topics are put into context with other topics from the course. Vægtning: The final examination counts 100% 7-trinsskala, ekstern censur | ||||||||||||||||
| Forudsætninger for indstilling til eksamen | 75 % of the reports must be approved in order to attend the final examen. | ||||||||||||||||
| Rammer for Undervisning | The course contains both a theoretical part and a practical part which are closely connected. There will be lectures as well as student presentations based on cases and journals. Laboratory work is running several days a week for 6-7 weeks | ||||||||||||||||
| Blokplacering | Block 3 Ugestruktur: Outside schedule | ||||||||||||||||
| Undervisningssprog | Engelsk | ||||||||||||||||
| Pointspærring | 240045 | ||||||||||||||||
| Anbefalede forudsætninger | 230003 240042 Tema: Eksperimentel molekylærbiologi 240019 Genome and Cell Biology | ||||||||||||||||
| Begrænset deltagerantal | Max. 42 studerents (lab facilities) | ||||||||||||||||
| Kursusindhold | |||||||||||||||||
| The course will contain a theoretical part where most aspects of peptide and protein production in biological organisms will be discussed (see below). You will acquire knowledge on the variety of possible host organisms found in the different kingdoms. We will discuss the possibilities of designing and finding new suitable expression hosts. Topics that will be covered in the theoretical part of the course: The intelligent choice of a host organism / Cloning strategies envisioned by an "in silico" multistep cloning / Promoter strength and induction / Copy number and silencing problems in heterologous hosts / Expression vectors / mRNA stability and introns / Choice of, and placement of purification tags / Stability of the product / Secretion of proteins and signal trapping / Post-translational modifications in different host organisms / Inclusion bodies and folding of proteins / Expression of membrane proteins compared to soluble proteins / Heterologous expression for production of antibodies / Expression of toxic proteins / Transient expression / Optimisation of expression level / Fermentation and large scale production. In the practical part of the course we will work with a range of different expression organisms. These include Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Xenopus oocytes and higher plants. We will transform them and determine the amount of produced protein, we will also discuss ways to optimize the expression level and finally prove that the expressed protein exhibit the desired function. In the practical part we will also cover a broad aspect of typical problems related to the production of recombinant protein. Topics from the practical part of the course: Expression and assembly of a multi subunit protein complex / The effect of alcohol and temperature on expression level / Expression of a secreted protein / Sub-cellular fractionation / Detection of post-translated modifications / The use of protein homologs from thermophilic bacteria /Yeast two-hybrid system/ Split-Ubiquitin system / Electrophysiological measurement on ion-channels/ Virus induced expression | |||||||||||||||||
| Undervisningsform | |||||||||||||||||
| The course contains both a theoretical part and a practical part. In the theoretical part there will be lectures as well as student presentations based on cases and journals. A practivcal laboratory part is running several days during most weeks. There is a close connection between the topics covered in the theoretical cases and the practical work. The course will be divided into smaller parts build upon the different expression organisms. | |||||||||||||||||
| Målbeskrivelse | |||||||||||||||||
| The production of technical enzymes as well as of peptide- and protein-based pharmaceuticals are in large scale being performed in specially designed host organisms. The aim of the course is to educate the students in processes associated with heterologous expression. The students will upon completion of this course be able to design and perform a strategy for the expression of a given gene. This includes considerations about amount, quality and downstream applications of the product. After completing the course the student should be able to: Knowledge: -Describe the main features of E.coli, Bacillus, S.cereviiae, P.pastoris, mammalian cell lines, Xenopus oocytes, Aspergillus and plants as expression hosts -Describe the following parameters for the above mentioned expression systems: Expression levels, Type of post-translationel modifications, Mechanisms for secretion of the product, Stability of the product, Stability of the transformed expression host, Methods commonly used for transformation, Strategies for optimization of the expression level and quality of the product. Skills: -Use the knowledge to design an appropriate strategy for the expression of the correct amount and quality of a given protein/peptide. -Design a strategy for creating an optimal genetically modified expression host in relation to reduction of proteases, improvement of secondary modifications and efficient compartmentation of the desired product. Competences: -Transfer theory and principles regarding the usefulness of different organisms as expression hosts to different work situations. -Make ethic considerations about the use of GM organisms for production of peptides and about the disease risks connected to a certain expression host. | |||||||||||||||||
| Litteraturhenvisninger | |||||||||||||||||
| The students will receive a collection of scientific papers together with the manuals for the laboratory part of the course. | |||||||||||||||||
| Kursusansvarlig | |||||||||||||||||
| Anja Thoe Fuglsang, atf@life.ku.dk, Institut for Plantebiologi og Bioteknologi/Sektion for Transportbiologi, Tlf: 353-32586 | |||||||||||||||||
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