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