Drug Effectiveness

Last Wednesday (December 19th, 2012),  I had my last internship in fall semester.

When I arrived, I met Eun Ji at the door, and she soon took me to the lab. Before we started we chatted for a while so we can know each other better. Eun Ji was a really approachable person. Although her English wasn't perfect, she was so amicable that I feel I could just talk to her on and on. She was relatively ne to Dr. Koffas' group and she was also a member of two other groups in RPI. Because she has a group meeting at 2, she assigned me a small task, and that was to  label all the chemicals, including where were they located in the lab in an excel file to help her work more efficiently. I was quite blown away by the variety of chemicals they had in the lab. There were over a hundred chemicals, not including those the lab people made them by  themselves, in the lab, and most of them I have never heard of. The names could be as simple as glucose and aluminum oxide to as complex as 3', 5'-dimethoxy-4'-hydroxyacetophenone, N,N'-Dicyclohexylcarbodiimide.

After Eun Ji was back, I followed her to the cell room where she showed me some cancer cells. I was so excited because I always wanted to see them! First, she showed me some normal skin cells. They were diamond shape and regular. Those cells are placed in a 96 wells plate where scientists could put a small portion of the cells in each well, give them different treatment, and compare the results together.

Then, I looked at the cancer cells, and not surprisingly, they grew irregularly. In this part of the lab, Eun Ji was testing the effectiveness of a potential drug. She divided the 96 well plates into 3 sections, each contained different kinds of cancer cells. Then, she divided each section into 3 smaller sections, each were giving different concentrations of drugs. Something like this:

She gave the cancer cells a specific dye (blue) that not only could stop them from growing but also make them easier for observation. Only the living cells would show the color blue, so after the cells were given treatments, we could know the effectiveness by observing how blue the well is (The bluer, the more living cells there were, the less effective the drug was).

Next, since all the living cells precipitated in the bottom of the wells, we could use a machine to suck out all the cell culture and dead cells, leaving only the blue living cells. Eun Ji then add another solution to the samples to wash off the blue dye. The solution would turn violet-pink as the dye was washed off. The samples was placed on a machine that works somehow like a cell culture roller that keeps a cell culture in motion in order for the cells to grow. The process would take about 30 min for all the dye to fully diffuse in the solution. The result follows the same principle: the darker the solution (more violet-pink color), the more living cells there are, so the less effective is the drug. After the dye comes off, we would put the plate in a spectrophotometry. A spectrophotometry works just like a spectrometer except it uses UV light instead of normal light, and the light transmits through 96 wells at once, so it gives us a more precise quantitative measurement of how many living cells there are.

While we were waiting for the dye to come off, we chatted a bit more. Eun Ji talked to me about the other 2 labs she was engaging in. One of them in which I thought was pretty interesting was that she is trying to find a drug that would prevent the fetus to get malaria if the mom has it. The drug would target the red blood cells that carry malaria parasites, blocking them before they transmit to the fetus through umbilical cord. As a biologist, Eun Ji said it was a challenge to her for that she now has to think in "chemistry way", deciding which molecule combing with which molecule would get the desired results. However, that's what science is about - not everything will yield the result you want, and you just have to keep trying. I found talking to her quite inspirational, and I shared my experience of talking about the Human Genome Project in class with her, too. Anyways, she told me that her newest drug has proved some effectiveness, and now she would need to find the right proportion of the formula so the drug won't kill the normal red blood cells as well. Working with Eun Ji was a very nice experience:)

The following two weeks are the winter vacation, so I won't be able to go to my internship. However, I am really excited when I come back:)

PCR Protocol

On Wednesday (Dec. 12, 2012), I did my safety training so I can get my own access ID to building. The training was online-based and pretty simple, and I ended up spending about 2 hours on it. I didn't get my ID right away because the person in charge was off that day. After my training, I went to Dr. Koffas, but since there was only about 40 min left, he didn't assign any project to me. At the last 10 min, he introduced me to his another post-doc, Eun Ji, whom I will be working with next week. It was such a coincidence because I had met her in the lab when I was with Namita the other day. She was very outgoing and nice. I also met few of Dr. Koffas' research group members too because they were going to have a group meeting. However, because I was running out of time, I couldn't join them. Nonetheless, I hope after I have gone to my internship for a few times, I can join several group meetings as Ms. Mroczka told me those group meetings are the most interesting part of a research. Anyways, although I didn't do much on that day, I was looking forward to working with Eun Ji and do some fun experiments:)

I would also like to briefly explain the PCR protocol, in which I read about last week. PCR (Polymerase Chain Reaction) is a common used technique to amplify a specific piece of DNA (to clone a strain of DNA). PCR involves in 3 processes, each requires a specific temperature:

After putting the prepared solution into a Thermal cycler...

1) Denaturing: Under 95℃, the double helix DNA was separated into 2 single strands.

2) Annealing: The temperature quickly cools down to 55-65℃, attaching two primers (forward primer and backward primer) to the end of each strand of DNA. A primers is a strand of nucleic acid that serves as a starting point for DNA synthesis and it is required because the enzymes that catalyze DNA replication, DNA polymerases, can only add new nucleotides to an existing strand of DNA.

3) Elongation: The temperature is then  increased to 72℃, catalyzing the polymerase synthesis of DNA.

Each 95℃-60℃-72℃ counts as a cycle, and the amplification usually runs about 30 cycles. By the end of the whole process, an single piece of DNA can be amplified up to a billion strands within just a few hours.

       Retrieved from Pray, L. (2008) The biotechnology revolution: 

PCR and the use of reverse transcriptase to 

clone expressed genes. Nature Education 1(1)

In this lab, we amplify the DNA that coded for proteins required by bacteria to synthesize NP. Then, we would connect the strand of DNA into a plasmid, a designed DNA in double-circle shape.

In the end, we insert the plasmid into the bacteria. In order to keep the foreign DNA inside bacteria, the scientists also include the DNA responsible for antibiotic-resistance in the plasmid and raise the bacteria in antibiotic environment, so the bacteria would need to keep the plamid in order to survive. (I think this was really clever!)

An Overview of Application of Technologies for Producing NPs

Today (Dec. 5th, 2012), I had my first full intern! I was a bit lost when I first arrived and made some effort to ind the building and luckily, I was able to follow one faculty to get into the building because I don't have my access ID yet.

After Dr. Koffas came back from his class, he introduced me to one of his student, Namita, who was very intelligent and nice. He suggested a plan that in those 3 weeks before the winter break, I will be working with a different student each time, and then I can choose which one to work with for the next semester. But before I went to the lab with Namita, Dr. Koffas gave me a small orientation around the building (so I won't get lost next time!). Then, we went to a lady's (which appeared to be the one whom I followed into the building!) office to ask about my access ID, and she set up a safety / tech training for me on my next visit. After that, we went back to Namita's office again and we are ready to kick off!

First, Namita led me to the lab. I was impressed by how many lab rooms the building has. There are hallways of labs, well-organized and each assigned to a research group. I was super excited because it was my first time to enter a real, intense lab, and I was fascinated by the abundance of lab equipment and materials they owned. I started out asking Namita with a very fundamental question: What is the whole lab about? What is the ultimate goal? After a long explanation and discussion, here is my summary (it's quite long so bear with me):


Goal: To use systems and synthetic biology to mass produce NP

NP stands for natural products, which are compounds produced by plants that are pharmaceutical and biotechnological importance. For example, they may be anti-cancer or protective against a certain disease.

1) Identify a specific compound in plant (that is known for having some therapeutic efficacy). The team usually gets the information about the plant from others (past) who had analyzed the plant but lacked the technology to identify the compound. Then, the team would purify the mixture (of the plant) to get one specific desired compound. Another way is to find compounds that give the plant certain properties or protect it against certain diseases, and we will assume the compounds would have the same effect on human.

2) In order to construct the synthetic pathway that yields the desired NP, we need to track back the synthesis of this NP in plant. In other words, what protein makes this NP? What gene codes for this          protein? And, what specific DNA is in this gene?

DNA --> Genes --> Proteins --> Products

The team then sequences the piece of DNA that is responsible for a specific protein production. DNA is made up by 4 main nucleotides (monomers) – A, T, C, G (excluding U for now). Different sequence of nucleotides makes up different DNA / genes that produce different amino acids (monomers for protein) Thus, by purifying a specific piece of DNA, we can eventually make the protein we want.

                         i.e. a sequence of DNA (single strain) : ATC AAT CGG TAT
                                                                    amino acid:  1 –  2 –  3 – …

3) After knowing the sequence of a piece of DNA, we would use PCR technique to amplify that piece of DNA in order to get a desired concentration.

4) Next, insert the DNA into bacteria so the bacteria can make and carry the protein that is used to synthesize NP.

• Because the synthesis of the product usually involves in many pathways and multiple steps, the team normally insert 3-5 pieces of DNA so the bacteria could make all the proteins needed in the pathway.

S1 --> P1 --> P2 --> P3 --> Product

• Then, we would add a specific substrate to the bacteria and the proteins the bacteria now carry can react with the substrate and mass produce the desired product.

substrate  ---protein--->  product

5) To insert the DNA into bacteria, we “shock”, or freeze the bacteria in the medium in -80℃ first so the pores on the membrane of bacteria would shut down. Next, take the culture out and leave it in 0℃ while putting the DNA in the culture. Then, put the culture in 40-50℃ water to shock the bacteria again so that those pores would open and take in the inserted DNA.

6) The scientists then give different treatment to the bacteria, designing a condition that will optimize the  production of NP from the bacteria :) 

One thing I think it is important as Namita had well-addressed is the advantages of using bacteria:

• easy to handle
• reproduced faster
• bacterial cells are less specialized, and thus, easier to take in foreign DNA and carry out the instructions more fully
• avoid ethical issue.

After the long explanation, she showed many different equipment in the lab and the techniques applied. For example, there is a machine similar to an advance spectrometer, and it can indirectly measure the number of cells in each of the 96 grids. There were also different fridges and cabins that stores / grow cells in different temperature. There were even specific test tubes / kits for different experiments!

At the end, we went back to her office, and she gave me the PCR (Polymerase Chain Reaction) protocol to read so I can do some experiments with her next time. I spend the last 40 minutes reading it, including discussion about the protocol.

Therefore, my assignment over this week would be to understand the PCR and hopefully I'll include the explanation of the protocol in my next post.

Although I didn't do any experiments today, I was pretty satisfied (and a bit overwhelmed) by the small lecture Namita gave me, and I am glad that I finally find a direction of where I am going:) I am looking forward to my next visit and to get my ID soon!

Here is an access to the article Dr. Koffas' team had recently published for detailed information (now it makes a lot more sense to me:D): http://dx.doi.org/10.1016/j.copbio.2012.08.010