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Protein Expression and Purification

The gene for Hfq (plus His tag) was inserted into the suffix region of BBa_K314103, a lac inducible expression cassette constructed by Washington iGEM 2010, and the entire construct was simultaneously inserted into the pSB1K3.m1 plasmid backbone using the Anza enzyme system protocol. See agarose gel below for confirmation of construct.

The resulting plasmid was purified and transformed into Rosetta 2 pLySs cells and grown in LB broth with a 30μg/mL concentration of kanamycin. Initially, 3mL of a 10mL starter culture was used to expand to a 300mL culture, which was grown until OD600 = 0.82 before being induced with 0.8mM IPTG. Cells were taken at t = 0h, 1h, 2h, 12h, 14h, and 18h, before being harvested after 18h. Protein expression levels over time are shown below in an SDS-PAGE gel below; protein expression levels appear to be highest at 18h (see SDS-PAGE gel below). Hfq was purified using a Ni-NTA column, and the resulting concentration in fraction 1 totaled 0.31mg/mL.