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Using high content analysis for the phenotypic visualization of neutral lipids in nitrogen-deprived Chlamydomonas reinhardtii
Author
Han, Bing Ling
Supervisor
Clarke, Neil
Lum, Shawn K. Y.
Abstract
The depletion of fossil fuels, coupled with the numerous environmental issues associated with their continued use, means that there is a growing and urgent need for alternative sources of energy. One renewable energy source that has been carefully scrutinized is biofuel. Algae offer a potential source of biomass for alternative fuel production and hold several key advantages over other organisms as possible feedstock for biofuels. They have the ability to synthesize and accumulate large amounts of neutral lipids, such as triacylglycerols (TAGs) which can be used as bio-diesel precursors. Algae can produce up to thirty times more energy per acre compared to crops like soybean, rapeseed or corn. They can thrive under harsh conditions, and on non-arable land so as not to impact on food production, and can utilize wastewater as a nutrient source. Chlamydomonas reinhardtii is a unicellular haploid motile green algae that is commonly found in soil and freshwater. Its genetic, molecular, physiological and genomic features have made it a model organism for the elucidation of many biological processes that are vital to both plants and animals, including flagellar function and photosynthesis. In this paper, high content analysis (HCA), or sometimes termed high content screening (HCS), technology is used to obtain information pertaining to the effects of nitrogen starvation on lipid metabolism of C. reinhardtii. The eventual objective of this work is to use this knowledge to help screen algae to be better feedstock for biofuels. HCA/S is a high throughput, automated cell biology technique that relies on fluorescent microscopy for information gathering, based on the detection of fluorescent tags, antibodies or dyes. It can be used to measure alterations in a wide range of cell characteristics like cell or organelle morphology, cell proliferation and protein translocation. C reinhardtii is ideal for use in a HCA system as it is easily cultured and can be stained with multiple dyes. Nile Red, a lipid-soluble fluorescent probe, has been successfully used in the detection of TAGs in several different classes of algae. It is hoped that the use of this probe within a HCA framework will offer a starting point for the rapid screening of algal cultures with perturbations in lipid production. In addition to screening lipid levels, other areas that will be explored here using HCA are lipid droplet formation and nuclear morphology.
At present, lipid metabolism in C. reinhardtii is a relatively unexplored area and little is known about the effects of knocking down lipid-related genes. A high throughput phenotypic screen for identifying interesting candidates for further investigation is a highly important component of any such study, and HCS offers an invaluable tool for any future research in this area.
At present, lipid metabolism in C. reinhardtii is a relatively unexplored area and little is known about the effects of knocking down lipid-related genes. A high throughput phenotypic screen for identifying interesting candidates for further investigation is a highly important component of any such study, and HCS offers an invaluable tool for any future research in this area.
Date Issued
2011
Call Number
QK569.C486 Han
Date Submitted
2011