Biological Chemistry A: Technologies for Directed Evolution of Proteins

Abstract

During this semester course, methodologies will be taught for biological-chemical enzyme evolution experiments using molecular genetic mutation technologies and in vivo selection in recombinant bacterial strains.

Objective

All technologies used for the experiments will be explained to the students in practice with the goal that they will be able to independently apply them for the course project and in future research endeavors. After the course, an individual report about the results obtained has to be prepared.

Content

This class conducts and supports experiments for a specifically designed genuine research project. We will carry out biological-chemical protein evolution experiments using molecular genetic mutation technologies and in vivo selection in recombinant bacterial strains. The relevant technologies will be taught to the students, such as the preparation of competent cells, production and isolation of DNA fragments, transformation of gene libraries, and DNA sequencing. The course participants will generate a variety of different variants of a cage-forming lumazine synthase. Individual capsid proteins will be purified and subsequently characterized using several different spectroscopic methods. The detailed chemical-physical analyses include the determination of their molecular mass and the integrity and stability of the protein structure. The cellular uptake of produced cages into mammalian cells will be assessed using confocal microscopy. The results obtained from the individual evolution experiments will be compared and presented in a final seminar and with a written report. We expect that during this lab course we will not only generate novel cage variants but also gain new insights into requirements for assembly and cellular delivery.

Resources