Methods: Approaching Our Project
As C. elegans is a multi-celleular organism, developing a concrete approach to our project required extensive research and planning. This section outlines our general methodology to tackling the bioremediation concept.
The Nose: Chemotaxis Mechanism
After narrowing down our project idea, the first step we took was to research compounds and G-protein coupled receptors (GPCRs) our worm could use to detect pollutants. Since our team consisted of students from across Canada, members of our team were aware of the issues surrounding the development of the Oil Sands in Western Canada, which was part of the rationale for the project. We therefore chose to work with GPCRs which bind chemicals present in pollutants such as those found in the Oil Sands.
Below are the GPCRs our team worked with, in BioBrick form.
Rattus novergicus-NPY1R_Rat
Mus musculus-NPY1R_Mus
Rattus novergicus-MARM1
Rattus novergicus-MARM
Homo sapiens-OX1R
The Nerves:
Since C. elegans is a multicellular organism, we needed to make sure our non-native GPCR was expressed in the neurons that would stimulate our worm to move towards the ligand. To accomplish this, we researched GPCRs that would normally be expressed in the olfactory (“smelling”) neurons of the worm. Then, we kept the promoter of the native GPCR and substituted the rest of the protein with our foreign GPCR. Hence, our constructs consist of a C. elegans promoter, followed by a non-native GPCR and terminated by the C. elegans UNC-54 terminator that our team researched last year.
Characterization
Our characterization process is shown below.
References
1. PharmacoInformatics Laboratory, GLIDA: GPCR Ligand Database (ver. 2.04, 2010), Kyoto University
2. Kiyohara H, Torigoe S, Kaida N, Asaki T, Iida, T, Hayashi H and Takizawa N. (1994) Cloning and Characterization of a Chromosomal Gene Cluster, pah, That Encodes the Upper Pathway for Phenanthrene and Naphthalene Utilization by Pseudomonas putida OUS82. Journal of Bacteriology, 176:2439-2443.
3. Ahmed S and Hodgkin J. (2000) MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans. Nature, 403(6766):149-151.
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