Jonathan Corbett approached me seeking to participate in a summer research project in my lab. I study leaf development genes in tobacco but it takes months to transform plant tissue and regenerate a transgenic plant. Students who work with me are generally limited to doing one small step of this process in a semester. Clearly this research could not be proposed for a summer project; something faster is needed. Last fall Kelsey Oleynek worked with me on an independent study project in molecular genetics that involved diagnostic cuts by restriction endonucleases of a GFP plasmid in E. coli that had been used in Organismal Biology instructional labs: pKScGFP. This plasmid has a version of the green fluorescent protein (GFP) gene that expresses strongly in bacteria, but has special codon edits for optimal expression in Chlamydomonas reinhardtii. This is a eukaryotic organism (unicellular alga) that can be transformed in a time-frame of weeks and the results analyzed within a summer project timeframe. As part of his preparation to do this kind of research, I have asked Jonathan to read articles, book chapters, and draft a proposal for this program. The following project description was drafted by him, but has been edited by me. I feel that this approach honors the spirit of the collaborative research concept of this grant program from its inception.
Genetic manipulation is one of the largest-growing fields of biotechnology in terms of both public and industrial interest. Transformation is a widely used process in which genetic information is moved between cells and subsequently expressed in the target organism. Chlamydomonas reinhardtii is a commonly-studied model organism in molecular research for several reasons, including inexpensive nutrient requirements, rapid reproductive cycles, and multiple available markers for each of its three genomes (nuclear, chondriome, plastome). Chlamydomonas is a non-pathogenic, resilient eukaryote, balancing cellular and structural complexity with laboratory convenience, and providing applicable experience in the fields of genetics and molecular biology.
The process involved in transferring genetic information from one organism to another involves several steps. A suitable gene must be placed within a DNA vector for transfer. The DNA must include an antibiotic resistance gene for selection of cells that have received it. For the proposed research, the DNA has already been designed specifically for transfer into C. reinhardtii and has been cloned into E. coli. We will prepare the host cells for receipt of new information by either removing or penetrating the cell wall, and the DNA will be linearized by endonuclease digestion and transferred into the live organism. Three common methods exist for transfer into Chlamydomonas: electroporation, silicon-carbide whisker-mediated, and glass bead-mediated transformation. All three methods are available at ECSU for this project and our first approach will be to use the one that is recommended by a colleague, Kriston Stipek, who is currently testing the relative efficiency of all three methods.
After a population of cells has potentially received the genes of interest, cells will be selected by placing the population in a medium with the eukaryotic antibiotic (zeosin in this case) for which the plasmid should have provided resistance. It is expected that only cells that have incorporated the plasmid into their genomes will survive. However, there is always the chance of false positive transformation. Therefore clonal lines of putative transformed cells will be analyzed for expression of the gene of interest.
The plasmid DNA, pSKcGFP, that we will be using features a gene for synthesis of Green Fluorescent Protein (GFP). When an organism successfully incorporates the GFP gene into its genome and expresses it, the cells fluoresce when exposed to ultraviolet light. Assuming successful transformation is achieved, we will try to determine the location within the cell where the cells have expressed these proteins via epifluorescence and scanning confocal microscopy. Detection of successful transformation of the gene for GFP and subsequent identification of expression sites within the cellÕs organelles is the ultimate goal of the proposed research.
The culture of both pSKcGFP::E. coli and C. reinhardtii, isolation and preparation of the plasmid DNA for transfer, and assembly of all necessary materials will be completed in May. By early June, transformations will be attempted and antibiotic selection for successful incorporation should allow for isolation of putative transformed cell lines toward the end of June and into July. Expression analysis and cytochemical localization should be achieved by the end of summer 2012. The findings will be incorporated into presentation materials for one of the 2012-2013 undergraduate research conferences.
Professor Ross Koning will facilitate and oversee all aspects of this project and will mentor the student in the laboratory techniques, cell culture, DNA transfer processes, and epifluorescence and confocal microscopy. He has extensive experience in these methods and basic training in the use of the confocal microscope. He will participate in the panel presentation. He recommends Jonathan Corbett to be selected as the student to collaborate in this research project.
Dr. Koning and Jonathan Corbett have worked closely together in laboratory primarily in Fall 2011 and Spring 2012 (with Jonathan as an outstanding Teaching Assistant) in Organismal Biology. Jonathan will have completed Organismal Biology, Cellular Biology, and Genetics laboratory coursework in our campus facilities by the projectÕs start. He also has experience in multiple laboratory courses in chemistry and biology both at ECSU and in California. He has collaborated in the design of this project and the writing of this proposal. Jonathan will be living in off-campus housing in Willimantic and has unrestricted access to reliable transportation. He is in good standing with the University, has served as a tutor in the MAC, maintains a 4.0 GPA, and is a member of Phi Theta Kappa National Honor society. Moreover, Jonathan has been accepted as the peer mentor for Dr. Koning's section of FYI 100 for Fall 2012 in light of his outstanding performance as a Teaching Assistant this semester and his interest in becoming a professional educator. If selected, and with supervision, Jonathan will participate in all laboratory work and data analysis, and will make the presentation at the student research conference/exhibition.
Most of the materials, equipment, and expertise are already available at ECSU for this project. The constructed plasmid containing both GFP and zeocin antibiotic resistance genes is already available as are cell lines of Chlamydomonas to be transformed. The equipment and reagents needed for this project include culture dishes, lighted shelves, pipettes, incubators, restriction enzymes, equipment for all three forms of DNA transfer including electroporators, and UV epifluorescence and scanning confocal fluorescence microscopes; these are already available at ECSU. Dr. Koning has been trained to use the confocal microscope and has years of experience with DNA manipulation in vitro and electroporation methods of transformation.
The primary expenses for this study include Zeocin, the antibiotic for which the resistance gene has been assembled within the plasmid of interest, and student summer stipend support.
Fresh Restriction Endonuclease $100
Zeocin Antibiotic 300 µg/mL required 1g vial $300
Student Stipend $3,100
Total Requested $3,500
Brown, L. E., S. L. Sprecher, and L. R. Keller. 1991. Introduction of exogenous DNA into Chlamydomonas reinhardtii by electroporation. Mol. Cell. Biol. 11: 2328-2332.
Dunahay, T. G. 1993. Transformation of Chlamydomonas reinhardtii with silicon carbide whiskers. Meth. Mol. Biol. 62: 503-509.
Franklin, S., B. Ngo, E. Efuet, and S. P. Mayfield. 2002. Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast. Plant J. 30(6): 733-744.
Fuhrmann, M., W. Oertel, and P. Hegemann. 1999. A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. Plant J. 19(3): 353-361.
Kozminski, K. G., K. A. Johnson, P. Forscher and J. L. Rosenbaum. 1993. A motility in the eukaryotic flagellum unrelated to flagellar beating. Proc. Natl. Acad. Sci. 90: 5519-5523.
Nelson, J., P. B. Savereide, and P. A. Lefebvre. 1994. The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation. Mol. Cell. Biol. 14: 4011-4019.
Stevens, D., R, S. Purton and J. D. Rochaix. 1996. The bacterial phleomycin resistance gene, ble, as a dominant selectable marker in Chlamydomonas. Molec. Gen. Genetics 251(1): 23-30.
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