목차
Flow Cytometry Based Enzyme Engineering
Enzyme enginneering을 이용한 세포측정법으로 the generation of fluorescence signal that reflects the phenotype of interest을 요구한다. Intracellular reaction, Cell surface display, Comparmentalization of whole cells, In vitro compartmentalization에서 이용된다.
1.Intracellular Reactions
Enzyme of interest : Expressed and maintained inracelluarlu Generation of fluorescent porducts in the cells by enzymatic reactons
2.Cell surface Display
Display of the enzyme on the surface of cells
Retention of fluorescent porducts on the cell surface by Georgiou and cowokers
3.Compartmentalization of Whole Cells
Rapid diffusion of products : critical probelm for the FACS based enzyme evolution
Compartmentalization of the cells in water-in-oil (w/o) emulsions : keep the phenotype in the compartment by Tawfik and cowokers
4.In Vitro Compartmentalization (IVC)
Aartificail cells, aqueous w/o or w/o/w emulsion
microcompartments or microdorplets
Coupled with in vitro transcription/translaton systems
Colony based Screens
Phenotypes affecting cell viability or differentiaton capability
Economical screening
Adjustment of screening stringency (chaning plasmid copy number, enzyme expression level or concretration of antibotics...etc)
Identified by naked eye (existence of colonies, size of colonies) or imaging system
Phage Based screens
Culture in deep well plates(작은 구멍에 enzyme을 expression 시키는 방법)
Storage of some amount of culture broth (glycerol stock)
Centrifugation
Lysis
Enzymatic assay
