The search for lignin degrading enzymes is starting to come to a close. After identifying which of our samples were producing enzymes using the guaiacol assay, we performed PCR on these samples and sent the isolated DNA of to be sequenced. After receiving the sequenced DNA, we blasted the samples using the GENBANK database. Our results were surprising. Apparently, the green mold we had thought was contamination was actually a LDE producing fungi! I guess we should have done some more research on the fungi we had assumed was hurting our samples before we jumped to conclusions.
Now that we have identified our samples, were are moving into the project assembly phase. Much of the past few weeks had been spent creating our final presentation, writing our paper, and creating graphs. With Christmas break coming and our project coming to close, our group is experiencing the bittersweet feeling of the end coming into sight.
Ryu and I have been working hard to align the final steps of our project. We made twenty four containers containing sterile dirt. We are then going to put different measurements of lead in each of them. After that we are going to introduce the two species of fungi that we found at Duke Energy. If the fungi grow we will then add earthworms into the soil to help them bioremediate the lead out of the soil.
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.
It’s always a good sign when you actually find what you’re searching for in an experiment, correct? Yes! After averting a possible crisis from contamination of a green mold, we finally (after weeks of plating and transferring, described in our previous blog) plated each of our isolated strains of fungi on guaiacol agars. We found the results that we’d been hoping for, three of our 27 samples have tested positive for the lignin degrading enzymes, ligninases. Soon, we will be able to perform PCR on our successful ligninase producing fungi and find out what we’re dealing with!
Testing! Testing! Aiden and I have been working hard this past month, and have made leaps in our project since our last update. Our fungi have been growing well and have be moved to new plates, this time spiked with lead(II). They have been growing for a week now, and are showing great results! This test is to see if our species of fungi can take up heavy metals, a critical step for a bio-remediation project. We used three different cultures of fungi, with five different levels of lead(II): 0mM, 1mM, 2mM, 3mM, and 4mM. We have observed in one group a yellow color has appeared! Lead(II) gives off a yellow color when reacting to other chemicals, so if the fungi is absorbing the lead or not, it is doing something with it. Now our next step is to find out what is going on. We are planning to regrow the fungi on a broth, an easy way to test for lead in the fungi itself.
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!
For the past week, we’ve spent nearly every day in the hood, plating fungi. Whether its isolating fungi from samples using water agar, or plating isolated fungi for growth on PDA, our time in TIME has been spent surrounded by the smell of agar and the hum of the hood’s blower. Due to our large number of samples and our limited time, its looks like this will continue to be a pattern for the time being. Everyday there will be things to plate, colonies to transfer, and assay’s to check. Let’s just hope there’s enough room in the incubator for all these stacks.
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!
Deep in the TIME room at Brevard High School, Ryan and Hannah have been submerging themselves into a new worldThe world of fungi, quite the fascination venture.
Their explorations are in the hot pursuit of local species of lignicolous fungi, or fungi that degrade lignin. Their studies of these extraordinary microorganisms have just begun, yet they have already been captivated by their preliminary findings dealing with fungi isolation. Who knew bleach would inhibit bacterial growth, but allow the fungi to propagate?!