Translocation of Cell-Penetrating Peptides into Emiliania huxleyi—Developing Novel Methods for Genome Editing in Algae

With recent advancements in genome sequencing and species characterization as a whole, the field of molecular biology has been revolutionized by the opportunity to investigate new organisms. While conventional biological systems and model organisms are well-characterized and well-known, researchers are finding that new organisms allow them to understand new mechanisms, uncover new molecules and make use of perpetually advancing technology. These opportunities, however, are often met with many obstacles, as the yearning to study more biological systems inherently requires new methods to be established—unique to each organism. Even though researchers may seek to study the synthesis and degradation of novel compounds in vivo, not all cells can be transformed and not all organisms can be controlled in a laboratory environment. For that reason, it is important for researchers to accurately identify potential species as model organisms and establish whether or not these biological systems can be investigated using conventional methods. If conventional methods do not exist for such an organism, then the new challenge becomes: can researchers feasibly develop new methods to transform and culture these organisms in a laboratory setting? Local research into one such species, Emiliania huxleyi, has proven fruitful in that a Cal State University, San Marcos laboratory has been able to sequence the entire genome of this small phytoplankton and identify many genes that may contribute to the novel compounds this organism can produce. Attempts to transform E. huxleyi with conventional methods, however, have prevented further investigation into this species, in that researchers are unable to inject plasmids for expression into the small cells. Researchers in Dr. Betsy Read’s lab have attempted to transform cells through methods of electroporation, sonoporation, chemical transfection and small-particle bombardment, however, all methods have been unsuccessful to date. In investigating new methods of transformation, I identified a similar problem in a related algal species known as Chlamydomonas reinhardtii, in which researchers used cell-penetrating peptides to translocate genetic material across the cell membrane. These small peptides, often referred to as CPPs, facilitate cellular uptake of genetic information and due to its success in Chlamydomonas species, I hypothesized that it may be successful in the transformation of E. huxleyi. Therefore, this SIR project sought to fulfill two specific aims: to establish and validate a protocol for E. huxleyi transformation using cell-penetrating peptides, and to optimize this protocol with a design of experiments that assessed the conditions for efficient and reproducible cell transformation of E. huxleyi. After successfully translocating CPPs labelled with FITC fluorescent tags into the cells, this project identified differential success of multiple CPPs, indicating that not all cell-penetrating peptides can be treated as equal.