Optimizing Micro-particle Bombardment Parameters for the Marine Algae Emiliania huxleyi
Emiliania huxleyi is numerically the most abundant coccolithophorid and is found throughout the world with the exception of polar sea waters (Marsh, 2003). The genome of E. huxleyi was sequenced in a collaboration between Dr. Betsy Read’s lab at California State University San Marcos and the U.S. Department of Energy (Read et al., 2013). The function of the vast majority of genes in the E. huxleyi genome is unknown. Being able to characterize these genes will give insight into the biological processes occurring in the cell. E. huxleyi transformation has not yet been successful which may be due to either the DNA delivery method or plasmid instability. For these reasons, the overall objective of this Semester in Residency (SIR) project is to develop successful DNA delivery procedures for E. huxleyi using fluorescently labeled oligonucleotides using a Design Of Experiment (DOE) approach. Micro-particle bombardment involves shooting gold microcarriers coated with DNA toward target cells. There are various parameters that can be manipulated with the aim of successfully introducing microparticles into E. huxleyi cells. Those tested herein include microcarrier size, helium pressure, gap distance, and target distance. Successful cell uptake was determined by analyzing cells after bombardment using flow cytometry. The one conclusion that can be drawn from both sets of bombardment experiments is that target distance is likely to have a statistically significant effect on bombardment using the Biolistics® PDS-1000/He Particle Delivery System (Bio-Rad Laboratories, 2012). Although it is still unknown whether increasing the target distance has a positive or negative effect on the bombardment process. The work carried out herein will pave the way for future studies aimed at developing a stable transformation system in E. huxleyi.