Team:Cornell/project/wetlab/chassis

Chassis

E. Coli

Escherichia coli is a gram-negative, facultative anaerobic, rod-shaped bacterium that is the most widely studied prokaryotic model organism for work with recombinant DNA. We are using DH5-α strains, which are electrocompetent and contain recA1 and endA1 mutations that increase insert stability and improve the quality of plasmid DNA prepared from minipreps. We are also using BL21 competent strains, which are designed for high-efficiency protein expression of any gene under control of the T7 bacteriophage promoter system. Under control of the lacUV5 promoter in its chromosomal DNA, T7 RNA Polymerase is expressed upon addition of isopropyl-1-thio-β-D-galactopyranoside (IPTG). Proteins under control of T7 promoter driven vectors are then expressed at high levels.The cells also contain the T7 lysozyme gene within the pLysS plasmid, which suppresses the activity of T7 RNA polymerase caused by leaky basal expression.