Recently, Jolizen has finally isolated an enzyme! We isolated cellobiase, an enzyme that breaks down a type of cellulose, We visited a local lab, called PharmAgra, that does pharmaceutical research. They have larger centrifuges available for our use. We took our fungus over after we added ammonium sulfate that was used to make the proteins a solid. After it was spun at 900 rotations per minute, the proteins all stuck to one side of the bottle. We tested the proteins, and they tested positive to be cellobiase! We could tell because when cellobiase is present, the solution turns yellow.
Eureka! We have found a method to culture our endophytes that works for different tests, and grows faster than on plates. A liquid shake culture is made by putting broth into a flask and putting it on a machine that rotates the cultures. This causes aeration which makes the endophytes grow faster! Now, Jolizen is also able to test for the presence of enzymes using a Biorad test. Once we detect the enzymes, we will use the shake cultures to start isolating the enzymes out of the fungi! Its a bit of a race to have everything cultured, and our enzyme isolation methods down pat before turkey day.
Over the past week, the Jolizen station has been quite colorful. Screening methods using dyes have been used to detect enzyme production. Originally, we were using dyed plates and looking for a change in color as the fungus grew on the plate over the period of one week or more. After unclear results with this type of method, a new idea for enzyme detection was adopted. After growing our fungus for a few days, the plates are then flooded with the dye and results are available in 10 minutes, rather than 1 week, and are much more definite. The first assay of this kind detected amylase, an enzyme produced in our saliva that breaks down starch. Our fungus was grown on potato dextrose and flooded with Gram’s iodine. The starch that had not been “digested” stained black, leaving a ring of clear fungal growth where our fungus had broken down the starch. Amylase is being produced! The second assay was stolen from Hannah and Ryan and was the answer to our prayers! Our fungus was grown on LDE plates. When we returned from the weekend, the fungus an the surrounding quarter inch were brown to orange in color while the rest of our plate was clear. This indicates the production of ligninolytic enzymes, or enzymes that break down lignin. Our last assay which detects for production of cellulases (they break down cellulose) will happen tomorrow. Our fungus has been growing on cellulose and will be flooded with Congo red dye. Let’s cross our fingers that it’s 3 for 3!
The pressure cooker has been running nonstop this past week in the TIME room, cooking every type of agar known to man. Our refrigerator is stocked full of potato dextrose, blue, red, and water agar, so come and get ’em! Yesterday and today, the infamous “Jolizen” team (made of members Eliza, Joe, and Lauren) has been transferring our fungus, Diaporthe sp., to plates filled with 3 different types of mediums. The red plates (making up the red stripes of our homeland’s flag) have cellulose and a special Congo Red dye (especially extracted and shipped to us on the back of African elephants straight from the heart of the Congo). If this dye shows discoloration by the start of next week, our fungus is producing some type of cellulase enzyme. We also are growing our fungus on blue plates (found in the blue star corner of the flag) which, if color change occurs, will indicate activity of an enzyme that breaks down lignin. Lastly, we cultured on potato dextrose agar (the white stars and stripes) for the purpose of maintaining a healthy base line fungus to take isolates from. We performed DNA extraction and PCR on these PDA cultures last week so that we can be sure they are still Diaporthe sp. Stay tuned for the results of our colorful tests!
Paper cultures, paper cultures, paper cultures… After a week of trying to make a working shredded paper culture, Lauren, Eliza, and Joe are trying something different. In the past, Jolizen has made all of their endophyte cultures on agar, but now they are moving on. Today, they are trying to nail down a method for making broth cultures containing lignin and cellulose. The eventual goal is to isolate enzymes. Enzymes are proteins that can help break things, like paper, down into simple sugars. If we are successful, we will be able to grow our endophytes on a paper culture along with an enzyme in order to make biofuel!
Now that project proposals are over, the real work begins! It is hectic and confusing in the TIME classroom because everyone is trying to figure out our first step. For Jolizen (Joe, Eliza, and Lauren), we are rolling in paper. This year, we are looking at growing endophytes, a type of fungus that mutualistically lives in and around the cell wall of plants, on a paper culture. In order to do this, we will use shredded paper to make a culture.