Laboratory/computer based Skills:
- Molecular Biology -all necessary skills to make constructs (including bicistronic constructs) and also site-directed mutagenesis. I have made many constructs to date using pGEM-T Easy, pET and pGex vectors.
- Protein biochemistry –covering protein over-production, purification, SDS-PAGE and native-PAGE analyses, affinity chromatography, ion exchange chromatography and gel filtration chromatography. I have experience in over-producing and purifying numerous difficult proteins by varying induction temperature, duration of induction and E. coli cell strains as well as investigating different buffer environments for proteins such as pH and salt concentration. I also have experience in over-producing soluble human protein in E. coli through gene sequence adaptation.
- Protein characterisation (functional, biophysical and structural) -use of bioinformatic tools (e.g. from Expasy and EBI websites), limited proteolysis, pull-down assays, ubiquitination assays, western blot analysis, gel filtration, co-expression, circular dichroism (CD), dynamic light scattering (DLS), isothermal titration calorimetry (ITC), Surface Plasmon Resonanace (Biacore), crystallisation screening, and X-ray crystallography (at various synchrotron sources; previously at SRS and presently at Diamond, ESRF and BSRF).
- Structure determination- I have experience preparing and freezing crystals, taking crystals to synchrotron sources, collecting datasets, solving and analysing structures using programs such as iMosflm, CCP4 based programs, CNS, Phenix, Coot and PyMoL. I have on many occasions collected native datasets as well as Single wavelength Anomalous Dispersion (SAD) or Multi-wavelength Anomalous Dispersion (MAD) datasets from Selenium labeled proteins and heavy metal soaked crystals.
- Bioinformatics analyses- use of necessary tools and software to analyse DNA/ protein sequences and protein structures, including those provided in Expasy, Ebi, NCBI webpages, as well as Phyre2, MEGA6, iToL, PhyoT, Chimera, LigPlot and PyMoL.