Project Report

Termites are unique organisms capable of digesting plant material. This ability gives termites extreme destructive power and makes them a key part of the nitrogen cycle. Termites digest plant material through a combination of their own enzymes and the actions of microbes living within their gut. This set of microbes, the termite gut microbiome, has evolved in the presence of a very specific environment. In particular, the termite gut microbiome is predicted to have adapted to the termite diet. Not all termites, however, have the same diet. Some termites are wood-eaters while others are soil-eaters. The bacterial group, TG3, comprises 10% of the termite microbiome. It is found in both wood-eating and soil-eating termites. How then has a diet of wood versus soil affected TG3 bacteria? In order to understand the specificity of TG3 to the termite gut, I undertook an attempt to obtain genome sequence for TG3 bacteria. TG3 are unfortunately unculturable bacteria and are naturally found in the presence of numerous other bacterial species in the termite gut. I therefore, attempted to use single cell genomics in order to 1) isolate single TG3 cells and 2) sequence the genome of a single TG3 cell. I conducted this work in Dr. Yuichi Hongoh’s lab at Tokyo Institute of Technology. Dr. Hongoh is a world expert in single cell genomics. I was provided with wood-eating termites (Nasutitermes takasagoensis), freshly collected from Okinawa (Figure 1). I dissected the guts from these termites (Figure 2). I then used a technique called laser dissection to isolate single TG3 cells (Figure 3). In this technique, termite gut material is spread onto a membrane and viewed under a microscope. The microscope is equipped with a laser capable of cutting the membrane. Using this microscope, I was able to direct the laser to cut out single TG3 cells. Single cells, however, do not have enough DNA for genome sequencing. I therefore used whole genome amplification in order to amplify all of a single TG3 cell’s DNA. Whole genome amplification is a simple reaction that makes use of an enzyme called a Phi29 DNA polymerase that is able to repeatedly copy a single cell’s genome to high enough copy for DNA sequencing. I succeeded in amplifying DNA from TG3 cells. After sequence confirmation, unfortunately, the DNA was found to not be from TG3 but rather from contaminating bacteria. My advisor and I believe this contaminating bacterial DNA was present in the Phi29 DNA polymerase itself. Since this enzyme binds DNA very tightly, it is difficult to acquire truly pure Phi29. While I was in Japan, I gave a one hour lecture on the research I conduct in America. I also attended an international conference in Japan and presented my work from my home institution there. At this conference, I was greeted by scientists I have met both in America and in Japan, including one who had just recently heard my talk in Japan! I spent many dinners meeting Japanese researchers. We discussed life in our respective countries and how science life differs in our two countries. In particular we talked about the lack of women in professorship positions in Japan and efforts being made to remedy this. Before leaving for my stay in Japan, I met with a group of high school students from Foster High in Tukwila, WA. This high school has been noted as being one of the most diverse schools in the entire nation. I gave these students an overview of my research project. I stayed in touch with these students via a blog (http://eatingforaquadrillion.blogspot.com/) over the summer. This blog was updated about once per week with details on the protocols and techniques I was learning as well as sights and my experiences in Japan. The blog became publicized at my home university and ended up attracting over 700 visits.

Agency
National Science Foundation (NSF)
Institute
Office of International and Integrative Activities (IIA)
Application #
1107536
Program Officer
Carter Kimsey
Project Start
Project End
Budget Start
2011-06-01
Budget End
2012-05-31
Support Year
Fiscal Year
2011
Total Cost
$5,748
Indirect Cost
Name
Dirienzi Sara C
Department
Type
DUNS #
City
Seattle
State
WA
Country
United States
Zip Code
98103