Site-Directed Mutangenisis

Yesterday (March 13, 2013), I worked with Namita on mutangenisis - a process by which the genetic information of an organism is changed in a stable manner, resulting in a mutation. She was trying to create mutants for VvSTS enzyme - an enzyme found in grapes family that helps produce resveratrol, an interesting compound I have written in my previous post (click here). She wanted to create point mutation, which only changes one amino acid sequence in a protein. However, a single change in amino acid sequence can change the shape of the entire enzyme and may result to increase enzyme activity / efficiency or the ability to uptake other molecules.

• Note: T197A = we want to change the threonine (T) at 197 site to alanine (A)

Procedure:

1)  Mutant strand Synthesis (by PCR) 

Attach a mutangenic primer to original DNA template. Use PfuUltra DNA ploymerase (high-fidelity and mutational tolerant) to extend primers, so the new synthesized plasmids are mutant.

2) Dpn I digestion 

Because DNA produced in organisms are usually methylated, Dpn I recognizes those original DNA (non-mutant) from E.coli. and digests them. The resulting mix only contains mutangenic plasmids.

3) Transformation 

Tranformed mutated plasmids in cells for them to produce mutant enzymes.

Retrieved from QuikChange II XL Site-Directed Mutagenesis Kit Instruction  Manual

We did PCR with T197I, T197A, and T197M and prepare a 30μl mix for each. While reviewing what I had to add, this time I focused more on techniques such as always check if the volume of solution in pipette looks correct, and vortex the materials to make sure the concentration is consistent before adding them to reaction mix.

While we were waiting for the PCR, Namita continued her protein extraction from a marine bacteria that she was working on with another student. The bacteria appeared purple because of a compound they were interested in (so pretty!). They repeated adding solvent and centrifuging the solution over and over because the compound was so hard to dissolve. Yet, eventually, by adding a lot more solvent, they successfully dissolve most of the compound:)

Where does this fit in the map?

I found mutangenesis super interesting, and I hope I can learn more about it in the future. I won't be going to my internship for the next two weeks due to the spring break. However, I am looking forward to what am I working on next!:)