Jonathan Neubert and Dr. Julianne Grose, Microbiology and Molecular Biology

Main Text

Nicotinamide adenine dinucleotide (NAD) is a crucial coenzyme used in the production of ATP and general cellular metabolism. Its structure consists of two nucleotides—pyridine and adenine—joined by a pyrophosphate bond. The pyridine allows the molecule to function as an electron carrier in the cell. In this capacity, NAD is involved in over 300 cellular reactions including enzymatic reactions in glycolysis, reactions within the TCA cycle, and especially in the electron transport chain (Grose et al, 2005).
Differing levels of NAD have been associated with many pathologies, including diabetes, inflammatory syndromes, neurodegeneration, and cancer (Magni et al., 2008). Current methods of measuring NAD levels in cells is rather insensitive, temperamental, and very time-consuming (Lundquist and Olivera, 1971). We have sought to provide researchers with a novel technique to easily detect NAD levels in vivo instead of current in vitro methods.

Because we hope to publish our findings in a scientific journal upon completion of the research I am unable to disclose details of the new technique. I would, however, like to provide a basic explanation, especially for those outside of the life sciences. When I started working on this project my understanding of microbiology and molecular biology techniques was rudimentary at best. I had learned of such techniques as polymerase chain reaction (PCR) and gel electrophoresis. However, it was not until I actually used these and other techniques that I realized why they were of such great importance.

A key aspect of our research involved the creation of various clones. Carefully-designed clones can be created to perform a variety of tasks. Cloning requires a researcher to take different segments of DNA and essentially fit them together anew. PCR is used to “amplify,” or make copies of necessary segments of DNA from other proteins, for instance. Specialized enzymes are then used to cut this DNA at known locations. Researchers then combine the various pieces of DNA by matching their cut ends, and allow an enzyme to ligate them, effectively gluing them together. This new strand of DNA will code for a protein which will be made by a cell’s organelle.

Working on this project has taught me how research can be both exciting and very difficult. Initially, I thought research was simply comprised of an idea that was followed with a set of procedures that would yield helpful results nearly every time. My naivety quickly dissolved. Dr. Grose was able to utilize her expertise in the field of cellular metabolism to design structures for the NAD biosensor. We set to work creating PCR fragments and constructing the clones. In a testing environment the finished clones did not give us the expected reading of NAD levels. Dr. Grose went back to the drawing board and designed a new series of constructs. We used the same procedure in the construction of the new clones, however their structures were quite different. After believing that we created the correct clones we sent them to be sequenced. The results from the sequencing center told us that there were large portions of the expected clones that weren’t present and other portions displayed high levels of uncertainty. Without positive results from sequencing we were forced to perform the cloning again. After re-trying the cloning process and experiencing great difficulty creating the correct clones, we have decided to place this project on temporary hold. Throughout this process I have learned how much work goes into research and how many opportunities exist for error. These errors, however, can be both learning experiences and the catalysts for new ideas.

We have decided to divert our energy to a new project that may greatly assist us in designing better clones for the biosensor project. As we investigate the unknown structure of a key protein in our biosensor, we will look specifically at how the protein is situated in different environments. We are currently restarting this project after an issue with the cell disrupter, a machine used to homogenize cells. We will be able to use a sedimentation machine at the University of Utah and are currently looking for a professor to help us crystallize the protein. These results will not only help us in the biosensor project, but will also produce publishable data.

I expect to continue work on these projects during the 2011 winter semester. I hope that during this time we will be able to better understand the structure of the protein. This knowledge will help us design and construct the biosensor. Upon completion of the research we hope to publish both the structural findings of the protein as well as the details of the biosensor project. We remain set on providing researchers with a method for real-time, in vivo measurement of NAD levels.

I have greatly enjoyed being able to work on the NAD biosensor project, and I thank ORCA for the generous grant that continues to help me dedicate time to lab work. I would also like to thank my mentor, Dr. Julianne Grose, for her patience with me while I navigate the learning curve of research. I recognize the unique opportunity I have to work so closely with a professor as an undergraduate and am grateful for the time and energy she places in these projects. This research has helped me both academically as I apply material learned in my science classes, and spiritually as I learn more about the complexities of creation.

Sources

• Grose JH, Bergthorsson U, Roth JR. Regulation of NAD synthesis by the trifunctional
  NadR protein of Salmonella enterica. J Bacteriol. 2005 Apr;187(8):2774-82.
• Lundquist R, Olivera BM. Pyridine nucleotide metabolism in Escherichia coli. Exponential growth. J Biol Chem. 1971 Feb 25;246(4):1107.
• Magni G, Orsomando G, Raffelli N, Ruggieri S. Enzymology of mammalian NAD metabolism in health and disease. Front Biosci. 2008 May 1;13:6135-54.