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?