Goal 1: To overexpress and purify two candidate proteins.
In order to express the protein of choice, the first step is Cloning. There are two parts to make a plasmid: a vector and the gene of interest. The plasmid with the gene of interest is transformed into E. coli with the molecular machinery to make the protein.
We use a simple, one day cloning method that we call LICKI Cloning.
The second step is expression of the protein. We were able to over-express one candidate on our own over the summer. With the help of the Protein Expression Facility at CSU (see acknowledgements), we over-expressed four more candidates by August.
Last, we have optimized protein purification by using a Nickel Column and Size Exclusion Column.
Goal 2: To order and purify the matching DNA for each candidate.
Initially, we ordered DNA from a company as a duplex. However, it was brought to our attention that the purity level for crystallization is critical to complex formation and crystal growth. Therefore, we used Dr. Shing Ho’s protocol for DNA purification. We have purified the original crystal DNA and the best insert DNA for three co-crystal candidates.
Goal 3: To crystallize the original co-crystals.
Before crystallization, the complex was formed by incubating pure DNA and protein together. This was verified by Native Gel Electrophoresis.
We have started screening for crystal hits via sitting-drop vapor diffusion.
Goal 4: To crystallize the ICC with a DNA insert.
We will pursue this goal in November and December after the Jamboree.