Amanda Rainwater

August 2021

Teaching high school science has opened up many doors for me that I never would have imagined possible when I started 22 years ago. I have taught internationally, organized and led overseas trips for students, put on the “leadership” hat as department head and a district TOSA (Teacher on Special Assignment) for two years, created curricula, taught professional development, presented at conferences and partnered with my alma mater (UW) to offer college courses to high school students. Four years ago, I had the exciting opportunity to open a new high school and develop a biotechnology program from scratch. I had worked in labs before, and loved my SEP experience at Fred Hutch, but after building and running this new program for several years, I felt as though I needed innovative and new ideas to help keep the program robust and cutting edge for my students. Thus, the Hutch Fellowship for Excellence in STEM Teaching was a perfect opportunity for me to keep up to date with current research practices and techniques that would allow me to help better prepare my students for their future educational and career choices.

My Hutch Fellows experience took place in Dr. Nina Salama’s lab, which focuses on studying the amazing world of the Helicobacter pylori, specifically its role in causing stomach ulcers and certain kinds of gastrointestinal cancers. One interesting aspect of this gram negative bacterium is its helical shape and how that shape plays a role in colonizing the lining of the stomach. The acidic and inhospitable environment of the stomach makes it very difficult for anything living to survive in it. Thus, in order to make its home there, the bacterium must quickly escape this caustic environment. Previous studies have shown that the helical shape allows the bacterium to “burrow” into the mucus lining of the stomach wall quickly (imagine the cork screw of a wine opener) in order to hide from the acidic environment and most of the immune system. You can read more about this here.

Click here for a video of Helicobacter pylori moving (Courtesy of A. Rainwater, Salama Lab, 2021).

Along with using mouse models and human cells to better understand H. pylori infections and cancer, specific genes are being studied in the lab to see how they impact the helical shape. Previous investigations have found that removing certain proteins in H. pylori drastically changes the shape (see image below). With the help of my lab mentor, Sophie Sichel and my lab partner, Maggie Lewis, we investigated the impact of two specific mutations in a cytoskeletal gene called ccmA (curved cell morph A gene) and how those might impact the structure of the protein produced and thus the overall shape of the cell. If the shape was altered, we hypothesized that this might also impact the ability of the bacterium to successfully infect the stomach lining.

The cytoskeletal protein CcmA is required for the helical shape of Helicobacter pylori. Our research focused on making two new mutants of the bacteria, one with a single point mutation and the other involved altering a short region of the gene responsible for the protein. With both of these mutations, we wanted to see whether it would impact the shape of the cell, and thus its ability to colonize effectively.

Mutating H. pylori to express our “crafted” proteins involved many steps and patience with myself as I was reminded of what it felt like to be a student again. Learning the importance of each step and why certain controls were needed in the “big picture” of our project allowed me to reflect on my own teaching practice with students and how challenging and exciting learning new information can be.

Our research process started with using a plasmid with the WT ccmA gene and an ampicillin resistance gene. This plasmid was mutated using a site directed mutagenesis kit with a primer that contained the desired mutation. Many copies of the mutated version were made using PCR and then transformed into E.coli cells in order to make sufficient amounts of the mutated plasmid. The plasmid was then purified from the E.coli cells and Sanger sequencing was performed to check for the desired mutation in the ccmA gene.

Once we verified the presence of the mutation, the next steps involved transferring the mutant ccmA gene to the H. pylori genome. In order to prepare the gene for this next step we needed to use a process known as PCR SOEing. This process involved adding sequences that are normally upstream and downstream of the ccmA gene in the H. pylori genome and adding it to our mutated ccmA gene. Once this process was completed and the “stitched together” piece (upstream + mutant ccmA gene + downstream) was purified, it was transferred directly to a colony of WT H. pylori. Upon receiving the mutated gene with the homologous regions, H. pylori naturally performed homologous recombination and “switched out” the catsacB gene for the mutated ccmA gene, thus incorporating it into its genome.

To verify that colonies had taken in the mutated ccmA gene, we used selective media to isolate them. The catsacB gene allows bacteria to grow in media with the antibiotic chloramphenicol, but not in the presence of sucrose. Thus, if the H. pylori had completed homologous recombination and switched out the catsacB gene for the ccmA gene, they should be able to grow on a sucrose plate, but not on one that has the antibiotic chloramphenicol.

Once we isolated our mutant H.pylori, we were now ready to view them and analyze their shape. Viewing them involved making slides, taking many digital photos and then using a technique called “thresholding”. Once we collected the thresholded images of our mutants and controls, we analyzed the images using Celltool to compare the side curvature and axis length of the cells to our controls.

Throughout this process, I was reminded of the critical value of controls in the scientific process. For example, we needed to validate that the CcmA protein from our mutated gene was actually being expressed by our H. pylori cells. In order to do this, we performed a Western Blot and indeed, our cells were expressing our mutated protein.

The mutations investigated during this summer experience did not progress at an equal pace, as the second one proved to be more challenging and required repeating many steps. It was a great reminder for me about the sometimes frustrating aspects of science and how repeated trials are needed. This story will definitely be shared often with my students when their own experiments don’t turn out as expected!

I am very grateful for this in-depth and rich research experience in the Salama Lab. I have learned so many valuable tools and information and am looking forward to incorporating it into my curricula and teaching. Thank you again to my amazing mentor, Sophie Sichel, Dr. Nina Salama, SEP and the rest of the Salama Lab for giving me this opportunity to enrich the quality of STEM teaching in my biotechnology program.