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!:)

Enzyme Activity Assay (Attempt)

On Wednesday (March 6th, 2013), Eun Ji and I caught up where we left last week and worked on enzyme activity assay.  Enzyme activity measures how much enzymes is present in a reaction and how active an enzyme is under certain conditions. Eun Ji showed me her initial result of the assay. She expected that the graph should look like that of the red line, with the reaction rate eventually level off when all the substrates are converted to products. Yet, instead, hers look like the blue line. 

Why did this happen? We still don't know. Yet, we have come up with some hypothesis:

• 3GT binds with the substrate instead of cyanidin Cl --> can't react
• either enzyme or the intermediate molecules denatured under the condition
• reverse reaction (The model below illustrates the enzyme action. E=enzyme, S = substrate, P=products. The main idea is that intermediate molecules can react reversely back to substrate while a small portion goes on to the second reaction to produce the final product.)

Enzyme assay examines the following control factors:

• salt concentration
• enzyme-substrate ratio
• pH
• inhibition (inhibitors decrease enzyme activity)
• activators (increase enzyme activity)
• temperature (most denatured in high temp.)

The two enzymes we examined are 3GT and ANS, and this time we examined the effect on enzyme activity under pH 6 and 7. We loaded our samples and some supplements in a 96-well plate. We spent quite a while recalculating the concentration because we had previously messed our calculation for the concentration of the standard. Also, the standard solution for some reason wouldn't dissolve until we diluted it to 10mM. Luckily, we recognized something wrong before we add everything and were able to solve them:) 

Division of the plate

Once all the substances were added, Eun Ji added HCl to the 0 min to stop the reaction (control), and removed them to 4 microtubes. The rest samples were put in the incubators. When it reached 15 min, Eun Ji would repeat the same thing she did with 0 min, and so on. Because we were short of time I wasn't able to see the complete process. Later Eun Ji would put this test tubes in a spectrophotometer to analyze the enzyme activity and construct a graph.

Since I didn't quite understand the whole process, the information above included some outside research. Nevertheless, I found some interesting facts about kuromanin Cl (the product). Kuromanin Cl belongs to anthocyans family. In a paper the Koffas group previously published, Anthocyanins are "red, purple, or blue plant pigments that belong to the family of polyphenolic compounds collectively called flavonoids". Their antioxidant properties give them the economic value in food dye.

Where does this fit one our map?

I will keep doing some research about the process, and I hope I can discuss this with my mentor next time!

Protein Purification

Last Wednesday (February 27, 2013), Eun Ji taught me the basic steps for protein purification. Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Eun Ji had already extracted the protein solution by breaking down E.coli. The whole process was done in a 4C room in order to prevent the proteins from degrading (so cold!!><) We went through the following steps to purify the protein:

1) Column Washing
We first set up two columns and pour some resin in them. Resin (ampiphiles) is a chemical used to attracted proteins to its hydrophobic region. The original resin contains buffer that may destroy the proteins, so we need to wash the resin by replacing the original buffer with our own buffer. 

Resin precipitated at the bottom because the particles are relatively big and close-knit. Resin lose its function once it is dry, so I help Eun Ji check them and add  more buffer every 20 min.

2) Loading Samples
Once the resin was fully washed, we load our protein mixture in the column. During the process, resin binds with the targeted proteins with its hydrophobic region while other proteins leaked out. One can repeat this step several times to make sure all the targeted proteins are bind to resin.

Resin can recognize the tag (red circles) we added to the targeted protein prior to this.

3) Washing
Keep adding buffer to the solution (at least 5x the volume of resin). This step can further purify the resin-protein mixture.

4) Elution
This step is very important. In order to separate the targeted protein from resin, we would add molecules (in this case maltose) that have stronger affinity that could bind with resin. This step would increase the volume of the solution.

5) Concentration + Column Regeneration
Last, we need to condense the solution by taking out the buffer. The resin can be re-used for several times.

Another important thing is that when we extract the proteins from E. coli., the mixture also contains proteinase that would degrade out protein. Thus, we need to add proteinase inhibitor to our buffer in order to prevent this.

Where does this fit in the big map?

Later Eun Ji would test the enzyme activity and determine what condition would maximize the production. She told me that the product she would get from her proteins (ANS and 3GT) reaction is currently used in food coloring (red) and food preservation. I thought I was interesting  that scientists suspected the molecule for being anti-cancer and anti-aging because of its anti-oxidant property!

Even though the process was quite long, and it was pretty cold in the room, I thought it was quite an experience!