Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Carmen Camarena - California State University San Marcos
Co-Author(s): Christopher Smith, California State University San Marcos; Betsy Read, California State University San Marcos
Emilania huxleyi (E. hux) is a groundbreaking marine phytoplankton that is efficient to grow in the lab and plays a novel role in the discovery of calcifying genes. Ranging from 3-5 microns in diameter, it has the potential to create calcium carbonate (CaCO3) shells which settle to the bottom of ocean floors and effect carbon levels in the ocean as well as carbon cycling in the environment. In 1983, E. hux was collected off the coast of Peru and separate samples were sent to culture collections in Plymouth, England and Bigelow, Maine. In time, the culture in Maine lost its ability to calcify while the culture in England did not. The two culture genomes were sequenced and it was found that 2 strains were identical, today known as 217 and 1516. Bisulfite sequencing was used to identify differentially methylated and differentially expressed genes within these two strains. The strain 217 is known to express calcification while the 1516 strain is known to express non-calcification. As isogenic lines (having the same DNA), the question is whether the loss of ability to calcify is related to epigenetic modifications to the DNA that has caused changes in gene expression. Epigenetic changes occur when chemical compounds are added to single genes to regulate their activity. These chemical compounds are found on or attached to DNA, however, they are not part of the DNA sequence. Epigenetic changes may determine whether genes are turned on or off and may influence the production of proteins in specific cells. Not only will identifying these calcifying and non-calcifying genes add data analysis to further study and regulate carbon cycling in the ocean and environment, but related organisms of its kind may also play a role in medical studies. For example, Octonia, which is also made of calcium carbonate, is found in the saccule and utricle regions of the human ear. When Octonia is disturbed or abnormal, individuals may be at risk of losing their balance and probable to injury. In this study, I aim to independently validate the differentially expressed genes between the strains 217 and 1516 using Real time PCR (RT-PCR). Specifically, I am interested in validating the over expressed and under methylated genes. To do this, both strains of E. hux were grown in culture and total RNA was extracted from the two. The RNA samples were run on a formaldehyde-treated agarose gel to confirm no DNA contamination and to confirm ribosomal bands. Further, cDNA was then synthesized and SYBR RT- PCR was performed. As I am in the first steps of RNA isolation, preliminary results from the RT-PCR are still needed to be conducted. If these genes are identified up and downstream of the genome, the data may be used to further research in identifying the exact functions of these genes and further studies can be done to analyze and regulate carbon fixation in marine environments while also leading research in regulation of atmospheric CO2.
Funder Acknowledgement(s): National Science Foundation
Faculty Advisor: Betsy Read, firstname.lastname@example.org
Role: Throughout the experiment I was responsible for making up solutions such as the extraction buffer, Hydrochloride, Sodium hydroxide, growth of cultures and extracting the RNA. I made the 10X MOP running buffer and I was also responsible for synthesizing the complementary DNA using specified primers. I wrote the script for the Biomek Real Time PCR machine and created the replicate samples for the PCR.