Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Sarah Carpe - University of Maryland, Baltimore County
Co-Author(s): Lian Jackson, University of Maryland, Baltimore County, Baltimore MD Stephen M. Miller, University of Maryland, Baltimore County, Baltimore MD Amrita Madabushi, Baltimore City Community College, Baltimore MD
Fossil fuels have an undeniable environmental impact and are becoming less readily available, leading to an increase in demand for sustainable alternatives that can be produced in large volume, while minimizing cost. Algal biofuels have potential to fill this demand. Since some algae can produce large amounts of neutral lipids that can be converted into biodiesel, they could potentially be more economic than ethanol from corn or other plant sources. Some advantages to algal biofuels include their ability to grow in various media, including freshwater, salt water, and even wastewater, their lack of need for arable land, and their genetic manipulation capabilities. The focus of much research on algal biofuels involves increasing biomass. This study uses the model algal biofuel organism Chlamydomonas reinhardtii, and focuses on an approach for increasing access to the limiting factor in algal biomass production, carbon dioxide. C. reihardtii evolved a strategy to increase CO2 in low carbon environments, called the carbon concentrating mechanism (CCM). The goal of this research is to increase a component of this mechanism, the low CO2 inducible protein (LCI1), responsible for transporting CO2 into the cell across the plasma membrane. We predict that if we can overexpress the gene encoding this protein, more CO2 will be transported into the cell, increasing the internal carbon concentration, and thereby increasing algal growth and biomass. First we used standard cloning methods to generate a nuclear expression vector that contains an LCI1 coding sequence flanked by C. reinhardtii 5’ and 3’ regulatory sequences (HSP70A-RBSC3 hybrid 5’ UTR + promoter and RBCS2 3’ UTR) and transformed it into C. reinhardtii, but we could not detect transgenic protein on western gels. Therefore we modified this construct by gene-synthesizing a bleomycin resistance gene fragment (ble; provides resistance to the antibiotic zeocin) with a 3’ viral 2A peptide sequence, and ligated it upstream of the LCI1 coding region in the original vector. Transformants that are resistant to zeocin should also produce the LCI1 protein because it is produced from the same transcript as the zeocin-resistance protein. We are now attempting to transform this construct into C. reinhardtii (strain CC503) via electroporation and transformants will be selected on plates containing zeocin. We will use western blot analysis to identify the best expressing transformants, and those will be compared to a wild type strain in an algal multicultivator to determine if LCI overexpression enhances growth. If the experimental strain shows an increase in growth rate, we can apply our methods to the biotechnology production organism, Chlorella vulgaris.
Funder Acknowledgement(s): These results were obtained as part of the Research Experience and Mentoring (REM) program in the Department of Biological Sciences at the University of Maryland Baltimore County. This program is funded by a grant (REM supplement to NSF-EFRI-1332344) from the National Foundation (NSF) Directorate for Engineering (ENG) Office of Emerging Frontiers in Research and Innovation (EFRI)
Faculty Advisor: Stephen Miller, email@example.com
Role: standard cloning methods, transformation into algae