Competent cells were thawed on ice until defrosted, and 30-40µl of cells were mixed with >50µg of DNA. After a 20 min incubation on ice the sample was heat shocked at 42 o C for 60 sec, and placed on ice for a further 5 min. LB broth or SOC medium was added to the cells in order to create a total volume of 1ml. Finally the samples were incubated for 60 min at 37 o C and then pipetted onto the relevant plate in volumes of 20µl, 200µl and 780µl and incubated overnight at 37 o C. Single colonies were then chosen from these plates and plated onto quarter plates which were incubated under the same conditions overnight.

Protein Expression:

Conical flasks or baffled flasks were inoculated with overnight liquid cultures. The appropriate antibiotic was added and the conical flasks were incubated at 30 o C at 180 rpm until an OD 600 reading of 0.4 was observed. IPTG was added to the conical flasks at a final concentration of 100 microM and flasks were incubated for 16 hours at 18 o C at 180 rpm. Cells were then harvested at 4000rpm and 4 o C for 20 min, the supernatant was discarded and the pellet frozen.

Protein purification:

Cell pellets were resuspended in nickel column binding buffer, sonicated and cell debris was removed via centrifugation at 20,000 rpm and 4 o C for 20 minutes. The supernatant was then applied to a nickel ion affinity chromatography column. The first 5 ml of flow-through from each wash step was collected and stored, the eluted protein was collected in 10 x 1ml fractions which were frozen for later use.

SDS-PAGE:

The 1 ml fractions from the elution step of the purification were defrosted and resolved on a reducing 12% SDS- PAGE gel. Samples that were analysed included whole cells pre-induction, whole cells post- induction, soluble cell fraction (i.e. initial supernatant), the unbound fraction washed out with binding buffer, as well as samples eluted during the wash and final elution steps. Elutions were then desalted in a spin column and run through a buffer exchange column. UV-visible absorption spectra were recorded on the final elution fractions that tested positive using Bradford's reagent, absorption maxima were expected at approximately 407 nm for proteins predicted to bind c-type haem cofactors.

Team:Kent/Experiments

Materials

Chemical Synthesis Materials:

Antibiotics for Plates:

Protein Induction Sugar:

Nickel Ion Affinity Chromatography Column Buffers:

Bacterial Transformation:

Protein Expression:

Protein purification:

SDS-PAGE: