One of this year’s High School Intern projects was focused on the common micro-algae known as Chlamydomonas reinhardtii, or “Chlamy” for short. Their goal was to increase lipid production in the algae through the revolutionary CRISPR/Cas9 system. To provide some background: algae grow and produce lipids when in nutrient-limited situations; however, the algae themselves do not grow. In normal conditions, the algae grow at a constant rate, but their lipid production is limited. Scientists are trying to find ways to keep algae growth constant while increasing lipid growth to get the most biomass to convert to biofuel. When they joined the ISB team at the beginning of the internship, they had varying degrees of experience with CRISPR/Cas9, Chlamydomonas reinhardtii, and wet lab procedures. Overall, their different experiences and prior knowledge balanced each other out, and made for a strong research partner pair. Pia, who had been previously researching the CRISPR/Cas9 complex, aided Katherine in finding research articles and using her own knowledge to further themselves in what they knew.
At the beginning of their internship, which spanned the course of 7 weeks, they were presented with a project that had 4 major steps. Although it was a lofty goal they wanted to achieve, they worked hard and got as far as electroporating the cells and plating them on the Paromomycin selective plates.
Of course, first step to answering their complicated research question was understanding how to answer it! Per the request of their mentors, Pia and Katherine spent their first week studying the ins-and-outs of Chlamydomonas reinhardtii, and how one might edit such an organism using the CRISPR/Cas9 system. The CRISPR/Cas9 system was originally found in bacteria and archaea as a substitute for an immune system. When a foreign invader attempts to infect one of these organisms, the CRISPR/Cas9 system will attach-to and cleave segments of the invaders DNA, and adding it to strands of the bacteria/archaea’s own DNA. This way, if the invader ever returns, the organism can make RNA that matches exactly to the invaders DNA, allowing it to bind to, and therefore deactivate the foreign invader. The CRISPR Cas9 system works in a similar when used for experimental application. The main difference is that instead of the organism obtaining the foreign DNA segments out of self defense, it is designed and provided to them by scientists based on which genes they desire to be cut.
The other significant difference is that in nature, the entirety of this process occurred in vivo, or in the cell body. While some of the reactions also occur in vivo in a scientific application, several steps must occur in vitro, or outside of the cell body. Whether it be in a test tube or a complex piece of machinery, dealing with DNA/RNA reactions outside of the organism can present some unique challenges. In this case, there is the challenge of transferring the DNA and RNA into the cell without killing it. One way to do this is through electroporation. Electroporation is a procedure that introduces small electric pulses to cells so that the cell wall opens up temporary pores, allowing material (the target DNA) to enter. It sounds easy enough, however, this procedure requires a great deal of balance. Not enough electricity, and the pores will not be wide enough to allow any DNA to pass through. Too much electricity, and the cells themselves will die. While reading through almost a dozen papers, Pia and Katherine discovered that there are several trends of what will offer the best possible chances of success. They took note of those trends and created a table of variables that they wished to test called a matrix. With this matrix, they performed 24 different trials with slightly varied test conditions to see which was the most efficient for electroporating cells.
In order to tell which was the most efficient, Pia and Katherine had to find a way to measure their results. Moreover, they would not want to use the Cas9 plasmid in an experiment that they are unsure will work, since it is rather expensive. Instead, they tested their electroporation matrix using a resistance gene to the antibiotic Paromomycin. This gene, if successfully added to the algae’s DNA through electroporation, would allow it to survive even in the presence of the antibiotic. To determine this, Pia and Katherine prepared agar gel plates treated with Paromomycin to plate their post-electroporated cells onto. If there is growth, that means the electroporation was successful and we now have plates to compare efficiency with in order to determine the most successful combination of variables! However, if the cells do not grow, that means something went wrong and the antibiotic resistance gene did not make it into the algae cells. In this case, Pia and Katherine would return to the drawing board to alter the variables they wish to test.
Once an optimal electroporation protocol is determined, the experiment can move on to part two, which is introducing the Cas 9 enzyme to the algae cells in hopes of knocking out a gene.
As well as having to advance in their project and balance research on the side, the two continued to accumulate experiences in the lab. Katherine, who had previously taken a microbiology class, knew her way around micropipets centrifuges, and knew how to run gels for electrophoresis, helped Pia throughout the lab in techniques and ways to complete procedures. During their time there, they aided Aza Allen, an undergraduate intern, in her various projects such as curriculum development and FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements). The two took cell samples of algae at in different environments over the course of their time at ISB, measuring Optical Density, Quantum Yield, pH, and cell densities. They learned how to read scientific papers, extract DNA, transform cells, plate cells, use sterile technique, use the autoclave, learned how to apply stoichiometry, measure data qualitatively and quantitatively, and use safety procedures.