High Throughput Microplate Screens for Directed Protein Evolution

Autor: Lori A. Rowe, Omar B. Alexander, Ichiro Matsumura, Melissa L. Geddie
Rok vydání: 2004
Předmět:
Popis: Publisher Summary This chapter examines protocols for a versatile but relatively inexpensive high throughput microplate enzyme assay. Escherichia coli cells constitutively expressing a library of proteins are seeded into liquid media contained in 384-well microplates. These plates are sealed and the microcultures are propagated to saturation. Clones exhibiting the most improvement over ancestral control cultures are isolated and used as templates for the next round of directed evolution. The seeding of transformed cells into microplate wells can be automated in stages. The transformed cells are aliquoted such that each well receives an average of one transformed cell. The Poisson distribution predicts that ∼ 37% of the wells will receive no cells, ∼37% will receive exactly one cell, and the remainder will receive two or more. An absorbance reading could be taken after overnight growth so that enzyme activity data can be corrected for cell density. The procedure for washing and sterilization of microplates is also elaborated.
Databáze: OpenAIRE