Mutagenesis: a. Explain NNN, NNK, and NDT degeneracies: what are they, and what are their advantages and disadvantages? b. When you change two codons with NNK degeneracy, how many colonies do you need to screen?

What is FACS, how does it work, what is its function in protein screening, and what limits the screening in directed evolution?

Describe a model for the evolution of enzymes in nature and describe how new functions can emerge.

What is substrate walking?

Give and explain one sequence-based method to increase stability.

Give and explain one structure-based method to increase stability.

Correlated positions: what are they, how do you detect/screen them, and what problems do they cause for enzyme engineering?

Explain the following terms:

• $K_m$
• Template for homologous models
• STRENDA
• Antagonistic epistasis
• Overfitting