Team:Utah State/Results

Text descriptions go here.

Text descriptions go here.

To demonstrate that the N-terminal 10x His Tag (BBa_K1162009) functions correctly, it was cloned in front of Green Florescent Protein (GFP)with the lac promoter+rbs system (BBa_K208010)to give the complete construct BBa_K1162013. This construct was expressed in E. coli grown in 50mL LB media with the addition of chloramphenicol and induced with IPTG. After allowing to grow overnight the cells were spun down and protein was purified with a nickel spin column (see protein purification protocol on protocols page). Fractions from this nickel spin column procedure were saved and run on an SDS-PAGE gel (see below). Since this was a GFP purification procedure, the different fractions were dotted on parafilm and place on a UV gel box to visualize the protein (see below).

From the SDS-PAGE gel it can be seen that there is pure GFP (~26.9 kDa) in the elution fraction number 2 and 3. The wash fractions do not appear to have any GFP which indicates that the N terminal 10x His Tag is strongly bound to the Nickel Column during washing steps. Coupled with the GFP dot image it is clear that this method of purification works as desired and adds another purification system to the registry.

After demonstration that the N-terminal purification system functioned as desired, other constructs were built to purify protein using this method.