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Master’s project in protein engineering by directed evolution
In this project we will use directed evolution, an approach in protein engineering, to engineer biotin carboxylase for high activity with free biotin, as well as a small number of biotin analogs. This is a key step in engineering biotin-dependent carboxylases to act on novel substrates.
Keywords: protein engineering, mutagenesis, directed evolution, biochemistry, enzyme assays, high-throughput, protein structures, enzymology
Protein engineering involves changing the amino acid sequence of an enzyme to improve its catalytic properties or to catalyze new reactions. Directed evolution is an approach in protein engineering where large libraries of gene variants are screened in several rounds, with the best variant serving as the parent for the next round. Biotin-dependent caboxylases are exciting enzymes as they can incorporate CO2 into biological molecules, a feat that few enzymes are capable of. Engineering these enzymes to work on substrates other than their natural ones is an important challenge. However, these enzymes rely on carboxybiotin as a CO2 donor. This intermediate is generated by biotin carboxylase. Naturally occurring biotin carboxylases display low activity with free biotin and therefore represent a limiting factor in the two-enzyme system.
Protein engineering involves changing the amino acid sequence of an enzyme to improve its catalytic properties or to catalyze new reactions. Directed evolution is an approach in protein engineering where large libraries of gene variants are screened in several rounds, with the best variant serving as the parent for the next round. Biotin-dependent caboxylases are exciting enzymes as they can incorporate CO2 into biological molecules, a feat that few enzymes are capable of. Engineering these enzymes to work on substrates other than their natural ones is an important challenge. However, these enzymes rely on carboxybiotin as a CO2 donor. This intermediate is generated by biotin carboxylase. Naturally occurring biotin carboxylases display low activity with free biotin and therefore represent a limiting factor in the two-enzyme system.
In this project we will use directed evolution to engineer biotin carboxylase for high activity with free biotin, as well as a small number of biotin analogs. This process entails generating libraries by site-directed or PCR-based mutagenesis, designing high-throughput enzyme assays, and screening the generated libraries for activity using those assays. Existing protein 3D structures will be used to interpret the effect of individual mutations.
You will learn the principles and experimental methods of directed evolution. You will gain an understanding of key quality-control steps necessary for the process. You will learn how to distinguish true positives from false positives, how to analyze the data generated and how to use that data for deciding with which variant to move forward in the next round.
In this project we will use directed evolution to engineer biotin carboxylase for high activity with free biotin, as well as a small number of biotin analogs. This process entails generating libraries by site-directed or PCR-based mutagenesis, designing high-throughput enzyme assays, and screening the generated libraries for activity using those assays. Existing protein 3D structures will be used to interpret the effect of individual mutations.
You will learn the principles and experimental methods of directed evolution. You will gain an understanding of key quality-control steps necessary for the process. You will learn how to distinguish true positives from false positives, how to analyze the data generated and how to use that data for deciding with which variant to move forward in the next round.
If you are interested please send me a letter of motivation at martin.engqvist@chalmers.se
Assistant Professor Martin Engqvist,
Chalmers University of Technology,
Department of Biology and Biological Engineering,
Division of Systems and Synthetic biology
If you are interested please send me a letter of motivation at martin.engqvist@chalmers.se
Assistant Professor Martin Engqvist, Chalmers University of Technology, Department of Biology and Biological Engineering, Division of Systems and Synthetic biology
Each year the IDEA League offers the students of its partner universities over 180 monthly grants for a short-term research exchange. In general, these grants are awarded based on academic merit. For more information visit http://idealeague.org/student-grant/