Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Charity Ensor - Baltimore City Community College
Co-Author(s): Nicholas Often, Baltimore City Community College; Lian Jackson, Baltimore City Community College; Amrita Madabushi, Baltimore City Community College; Stephen M. Miller, University, Maryland, Baltimore County
There is need for clean alternatives to fossil fuels and green algae have great potential in this regard as sources of renewable liquid transportation fuels. Green algae are photosynthetic organisms that can be induced to produce relatively large amounts of neutral lipids that can be easily converted into biodiesel. The unicellular alga Chlamydomonas reinhardtii is the major model organism for biofuel production research as it is easy to genetically modify and it grows rapidly. This work focuses on increasing carbon dioxide (CO2) uptake as part of an effort to increase Calvin cycle function and algal growth rates. The carbonic anhdyrase 3 (CAH3) gene encodes an enzyme that converts carbonate into CO2 in the pyrenoid for carbon fixation by the enzyme rubisco, and we are testing the idea that overexpression of this enzyme should improve cell growth. We generated a nuclear expression vector that contains CAH3 coding sequence flanked by C. reinhardtii 5’ and 3’ regulatory sequences (HSP70A-RBSC3 hybrid 5’ UTR + promoter and RBCS2 3’ UTR) and transformed into C. reinhardtii, but we could not detect CAH3 protein in any of the transformants. To improve expression, we gene synthesized a bleomycin resistance gene fragment (ble) with a 3’ viral 2A peptide sequence and are inserting this fragment just upstream the CAH3 coding region in our CAH3 vector. Bleomycin-resistant transformants should also express CAH3 because ble expression is coupled to expression of the downstream gene (CAH3). Western-blot analysis will be used to determine accumulation of CAH3, and expressing transformants will be grown in an algal multi-cultivator to analyze growth rate of the transgenic algae compared to a wild type strain. If overexpression of CAH3 improves C. reinhardtii growth, the next step will be to attempt overexpression of this enzyme in production algae such as Chlorella vulgaris.Abstract CAH3 Charity Ensor final.docx
Funder Acknowledgement(s): NSF
Faculty Advisor: Stephen Miller, email@example.com
Role: I cultured E. coli with CAH3 gene and E. coli with bleomycin+2A peptide fragment, individually. I inoculated media in tubes to grow out single colonies of each. I cleaned up the DNA from each using multiple techniques including a DNA purification kit, mini boiling preps, and ethanol precipitates. I used an enzyme, Xho1, for a restriction digest of CAH3 vector. I ran diagnostics using gel electrophoresis, and completed multiple ligation techniques.