End of the Year Poster Section

Today (May, 1st 2013) was my last day of internship of the year! Time passed by so fast and I couldn't believe I have come so far! I made aposter and we interns presented our research during lunch today in Kellas Commons. Many people came and we explained our researches to them. Not until I repeated myself over and over again did I realize how much I actually learned. What seemed so incomprehensible back in November now made perfect sense to me:)

A screenshot of my final presentation poster.

That afternoon I went to RPI for the one last time. Eun Ji gave me a general view of some cool stuff she was working on in other labs. Guess what are those?

Frozen bovine (cow) cornea!

On the project on malaria prevention in fetus during pregnancy, Eun Ji was trying to extract and modify a compound from bovine cornea that could bind to specific receptors on placenta to block parasite-encoded variant surface antigens to bind to those receptors and transmit malaria to fetus. Later, I also helped prepare some dye for silver staining that is used widely in protein detection in gel.

In the end, I said goodbye to both Eun Ji and Namita. I really had a wonderful year doing STEM internship at RPI! From the people I worked with, to lab environment, to the actual lab work, it gave me a taste of the life of a scientist. The uppermost thing I would take away from this experience in addition to a plethora of laboratory techniques is the “qualities of a scientist” – precise, robust, patient, inquisitive, inventive, and inspirational. Not everything will yield the result we want, and we just have to keep trying. This experience makes me more certain about my goals. In the future, I would like to participate in more research focusing on molecular biology to enrich my experience and widen my horizon! Last but not the least, I will be working in another lab at Cornell this summer on intracellular communication, which I am very excited about! I will keep posting interesting things and events, so stay tuned!:)

When I grow up...

Happy National DNA Day!

This Thursday April 25, 2013 was the National DNA Day!!! This year particularly is the 60th anniversary of Watson & Cricks' discovery of DNA structure and the 10th anniversary of Human Genome Project (HGP)!

Retrieved from ASHG

I am especially interested in genetics because of its intricacy and roles in diseases. HGP provides scientists an unprecedented opportunity to better understand the role of genetics in human health and gradually reveal the mystery of genetic diseases. Today, genetics becomes increasingly important in diagnosis, drug development, and new treatments. Bellow is an excerpt of an essay I wrote on HGP:

HGP, though it does not boost the speed, increases the “success rate” of drug development. Identifying a specific mutation allows scientists to develop a targeted drug that directly tackles that mutation. Many studies on targeted drugs are done in cancers. Targeted cancer therapies are drugs or substances that inhibit the uncontrollable growth of cancer cells by blocking growth factors or inducing cell death (apoptosis). HGP facilitates the identification of these “targets”, which are usually defective genes that encode for proteins involved in cell signaling pathways. For instance, in chronic myeloid leukemia (CML), researchers had identified gene BCR-ABL – a result of translocation between chromosome 9 and 22. This gene produces a hyperactive protein that keeps Abl signaling pathway active and causes continuous proliferation of CML cells. Researchers can then develop a drug that represses this defective gene and treat the deadly disease (National Cancer Institute, 2011).

Here is the speech delivered by Francis Collins, the director of NIH, on National DNA day: http://directorsblog.nih.gov/dnas-double-anniversary/#more-1194 I really I could attend the annual ASHG meeting someday! Anyways, Happy National DNA Day!!

Because I had proctor training this Wednesday, I wasn't able to go to RPI. My internship is soon coming to an end, and I have working on my poster for my presentation:) I hope it'll all go well!

High-Performance Liquid Chromatography (HPLC)

Last Wednesday (April 11th, 2013), Eun Ji showed me how a high-performance liquid chromatography (HPLC) work. A HPLC is a chromatographic technique used to separate a mixture of compounds in biochemistry or analytical chemistry to identify, quantify or purify the individual components of the mixture [1]. 

Simplified map of a HPLC
Retrieved from http://web.nmsu.edu/~kburke/Instrumentation/Waters_HPLC_MS_TitlePg.html

First, we connected the computer to the machine. We can control the flow rate by typing in the computer. Eun Ji had made two kinds of buffer (solvent) A and B for her proteins, each of which had a thin tube connected to the pump where the two buffers mix. We typed in 1.000 ml/min for the flow rate (*A+B = 1ml not 1ml for A and 1ml for B) Overtime, the concentration of A decreases while the concentration of B increased, but the flow rate remained the same. This means that [A] and [B] in the mix solution in constantly changing. Concentration of A and B manipulated the polarity in the column.

Next, Eun Ji injected her sample through injector. 

Together, the solution and sample traveled to the column. A column contained very small resins that formed a fine filtrate. The sample contained a mixture of proteins. However, according to the affinity of each protein, proteins gradually separated from each other as [A] and [B] changed and flew out the column at different times.

Black sample is separated into blue, red, and yellow (3 proteins)
Retrieved from http://www.waters.com

As the separated protein bands leave the column, they pass immediately into the detector. The computer then construct a graph that contained "peaks" in it. Each peak represented a protein, so by counting how many peaks were there, we were able to determine how many kinds of protein were present in the sample (*but cannot determine "what" proteins are they unless by using special HPLC or conducting further study).

Retrieved from http://www.waters.com

However, we there were something wrong with the machine when we ran our HPLC. The pressure of the tubes continued to rise (normal: A-60, B-100; ours: A-90+, B-140+) and that the pumps automatically stopped to prevent the tubes from bursting. We reset the machine again, but it did not help. In addition, pump B was making weird noise, so we stopped our experiment. Nevertheless, I thought HPLC is a really brilliant tool. Before I have read many papers containing HPLC in their methods, and I am very glad that I now know what it means!

Where does this fit on our map?

Buffer for Protein Purification

This Wednesday (April 17, 2013) I prepared for some buffers for protein purification with Namita. We made four 200 ml buffers:

1. Lysis buffer - used to lyse the bacteria in order to collect the proteins. The solution would contain all proteins the bacteria produce. 
2. Wash buffer (20mM) - used to wash out some undesired proteins
3. Wash buffer (150mM) - further wash out the undesired proteins by breaking bonds between undesired proteins and resins
4. 300mM - 300mM imidazole solution can break the bonds between the targeted proteins and Ni. After washing away other proteins, the column by this time contains mainly the targeted protein bonded with nickel. 

Materials

• H2O
• NaH2PO4
• NaCl
• Imidazole 

The buffers only differ in their percentage of imidazole. Imidazole is used to separate bonds between nickel and proteins. The targeted protein is his-tagged, which have a high affinity to nickel when running through the column. However, some random proteins can also loosely bind to Ni too. The higher the concentration of imidazole, the stronger bond it can break. Here 300mM is the concentration to break the bond between our proteins and Ni.

To determine what concentration breaks the bond between targeted protein and Ni, one runs a gel to determine the size of the target protein and at what [mM] does the solution contains the most targeted proteins.

Example of a protein gel.
Retrieved from http://www.sciencedirect.com/science/article/pii/S0168165610001926

For example, as shown above, if lane 1 is 10mM and increases by 10mM each lane. At 80mM (lane 8), a clear dark band is shown, meaning that 80mM of imidazole breaks the bond between OmpA70 and Ni the best. Othe lower concentrations are used to washed off some contamination shown by the blurring bands in lane 1-7.

After we added the appropriate amount of substances into four flasks according to the calculation, we need to make the pH into 8. We do this by adding NaOH to the solution and using a pH meter to measure the pH.

pH meter (right) and magnetic stirrer (left)

It took us quite a long time though, especially the two with higher concentration of imidazole bacause imidazole is slightly acidic. Luckily, we have the magnetic stirrer, which i thought was a very brillant invention, to speed up the mixing rate.

Magnetic stirrer

When all the buffers finally reach pH8, we decided to call that for a day. Making buffers, as Namita admitted, can be very boring, yet it is very demanding because everything should be very concise. Later, Namita would use those buffer to establish a  imidazole gradient to collect and purify the proteins from E.coli. For the details please look here.

Where does this fit on our map?

P.S. I will post the blog from last week asap!

Mutangenesis (Continued)

On Wednsday (April 3rd, 2013), I continued working on mutangenesis in VvSTS enzyme with Namita. After we created several mutant plasmids by PCR last time, she added Dpn I to the solution to digest the original non-mutant DNA. Then, she did a biotransformation with a strain of bacteria (BW27784) that has the ability to ligate the new plasmids (because the polymerases actually form open-ended plasmids in PCR). Later, she sequenced a few transformed colonies in order to make sure that she had the right mutant before she did another transformation with another strain of bacteria for expression. In other words, the first transformation was to complete the circular plasmids and to check if the mutant plasmids have the right sequences. The second transformation was actually for cell expression.

Now came my task of the day: to create stock of mutant E.coli. There are 6 samples in total: control, T197I, T197A, T197M, et. all.

Namita provided six 15ml tubes. Since aeration is important in cell growth, a container can usually only holds 1/5 of its max. volume of solution. I put 3ml of cell media (3/15 = 1/5) and 3μl of antibiotics (1μg / ml) to each tube. Adding antibiotics is essential in making sure that the cells keep the mutant plasmids, which contain antibiotic resistant gene. Then, I pick one colony from each plate and add it to the media. Lastly, we put the tubes in incubator and let the cells grow a day, and then store them in -80C.

My job today was short and simple. Yet, precision was very important so I had to be careful in every step. I am looking forward to further discuss with Namita about the process:)

Where does this fit on our map?

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!