Overexpression of CAH1 and CAH8 in Chlamydomonas Reinhardtii

Undergraduate #37
Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology

Binika Chunara - Baltimore City Community College

Lipids derived from algae hold great promise as a sustainable fuel source. However, commercial production of algal fuels is expensive in comparison with fossil fuel. Genetic manipulation of algae can make biofuel production more efficient. The photosynthetic green alga Chlamydomonas reinhardtii is a wellstudied model organism that is easily manipulated at the molecular genetic level. In this study we are focusing on improving the carbon concentrating mechanism (CCM), a biological adaption to low carbon dioxide levels in the atmosphere.

The CCM is composed of mainly three components: carbonic anhydrases, CO2 transport proteins, and the pyrenoid. Carbonic anhydrases catalyze the interconversion of carbon dioxide and bicarbonate and thereby make inorganic carbon more accessible to the cell. The aim of this study is to increase the uptake of CO2 in C. reinhardtii by overexpressing two periplasmic (between the cell wall and membrane) carbonic anhydrases, CAH1 and CAH8, and ultimately determining the effect on growth rate. C. reinhardtii CAH1 and CAH8 coding regions were synthesized with C. reinhardtii codon bias and epitope tags and the gene fragments were subcloned into expression vector pARG which contains the ARG7 gene required for arginine biosynthesis. We transformed the CAH1 vector into an arg7 mutant strain and selected several ARG+ survivors for western blot analysis to determine the expression of protein; CAH8 lines will be generated later. We will select the best expressing lines for growth curve and dry weight analysis to determine whether the transformants overexpressing CAH1 or CAH8 are able to grow faster than the wild-type C. reinhardtii strain. In the future both genes could be expressed together. If we are successful, the next step will be to incorporate these methods for microalgae that naturally produce higher lipid levels than C. reinhardtii, but are harder to manipulate, such as Chlorella vulgaris.

Funder Acknowledgement(s): This program is funded by a grant (REM supplement to NSF-EFRI-1332344) from the National Science Foundation (NSF) Directorate for Engineering (ENG) Office of Emerging Frontiers in Research and Innovation (EFRI).

Faculty Advisor: Stephen Miller,