Category Archives: Students

Microbe vs. Microbe: My experience at CAZyme lab

It was the spring semester of my Masters programme at Stockholm University when I first contacted Lauren McKee to ask about a potential Masters thesis. From the very beginning, she was welcoming and talked with me at length about the possible projects that I could do for my thesis.  Following our meeting, I had a lecture on “Microbial interactions in the rhizosphere“ from Lauren. A particularly interesting concept to me was manipulating the behaviour of a biocontrol agent (BCA) to perform better in terms of increased growth or increased production of anti-pathogenic enzymes. This manipulation can be achieved by providing carbon sources specific to both the BCA and the plant pathogen. And that is where most commercial biocontrol formulations go wrong. Failing to understand how and what a BCA responds to leads to inefficiency. One of the important research themes at the CAZyme lab is to elucidate the molecular mechanism of nutrient-microbiota interactions: this will help us to create more effective and science-based solutions for sustainable agriculture. In this photo you can see me setting up one of the dozens of six-well agar plates I used during my thesis project!

I decided to work on a 3-week course project to familiarise myself with the ongoing work within Lauren’s group. In this project, I looked at the biocontrol properties of a gram-positive bacterium Bacillus subtilis natto against plant pathogenic oomycetes – Aphanomyces euteiches and Aphanomyces cochlioides – based on a previous thesis and publication from Anna Schönbichler, a former student in the group. During this period, I got to interact with CAZyme group members and other research groups within the Division. The best part is that it was a multicultural environment in the lab and it has been amazing getting to know people from many different backgrounds. 

Working with plant pathogens, in terms of plant protection, has always interested me and I believed that this would be a great opportunity for me. I started my year-long thesis in September 2020 and I worked on the topic “Assessment of Bacillus subtilis natto as a potential biocontrol agent against Aphanomyces euteiches,” which was a continuation of my earlier short project. For my thesis, I grew the bacterium in a variety of carbon sources that were representative of the cell-wall of the oomycete pathogen. This was done in order to gain an understanding of how the bacterium will respond when faced with the “real” pathogen. Following this, the enzymes and proteins produced by the bacterium in response to these carbon sources were analysed for cell wall-degrading activities and their ability to inhibit pathogen growth on an agar plate. This was really a fundamental investigation into bacterial biochemistry. Being a plant biologist, I wanted to understand the dynamics of the relationship between the host plant, pathogen, and biocontrol agent. For this, experiments using pea plants were conducted in growth chambers in a collaborator’s lab in Uppsala. It was found that there was some inhibition when a certain carbon source was used to manipulate B. subtilis natto. But, sufficient data was not produced to conclusively say that this bacterium could potentially be used as a BCA against this particular pathogen. Due to the natural variability in how plants grow, and how they respond to a pathogen, more replicates than I could study in my thesis would likely be required in order to generate statistically meaningful data. However, I believe that this project will serve as a blueprint for further experiments using B. subtilis in combination with other plant pathogens.

The research in Lauren’s group on biocontrol using soil bacteria is absolutely relevant for both the present and future world scenarios. I have to mention that the entire time I spent in the Division of Glycoscience with Lauren and other members was a fantastic learning phase. As a newbie researcher, I made mistakes (quite a few!), but I learnt from them and I couldn’t have asked for a better learning experience. I was able to work very independently within the group, so I figured out how to think ahead and manage my own time. Lauren was very encouraging and seldom lost her patience with me. Throughout my project, I learned better ways to communicate, plan, and truly understand the impact of science in everyday life. Scientific research is infinite, like human stupidity (sic). I have talked to other people working on biocontrol research and they tell me a similar story: there is always one small detail that is left to be discovered. I hope that future group members will continue with this project, avoid making the mistakes I made, optimise, and discover more and more. 

A Summer at the Stockholm CAZyme Lab – Insights and Experiences

As students, there are definitely times when it is hard to see how all of the knowledge that you are gathering during your study period can be applied in reality. When you are getting towards the end of your studies, you can definitely start to feel confused about what you actually want to work with after graduating. It is difficult to choose a certain career path when it is not clear what the roads are that lie in front of you. As we are currently still master’s students, we are still eager to learn and get new experience. Therefore, this was a perfect time and opportunity for a full-time summer internship at KTH. We are Elin and Srijani, and here we will share our experience of summer work at the Division of Glycoscience.

We undertook a seven-week internship in Lauren McKee’s group at the laboratory, where both of us were working on different projects under different supervisors; He Li and Mengshu Hao. Elin was helping He Li with her post doctoral project on biomass hydrolysis, and Srijani was working with Mengshu on a project on sustainable polysaccharide hydrogel formation. Both of us mainly focused our work on protein production and purification, and further testing these proteins in necessary assays. Srijani was performing an assay named Pull Down Assay, which basically checked the potential for ligand-binding and hydrogel formation at room temperature. Elin was working on optimizing the production and purification of a few proteins, and also tried some temperature optimization in assays of the purified enzymes.

What could a work week look like? The week usually started with preparation for protein purification, which meant that we had to cultivate the over-expressing bacteria and everything that comes along with that. For instance, these preparations could be making litres of growth media, autoclaving culture flasks and as many pipette tips as you can fit in the autoclave, booking instruments/machines that are needed, etc. The proteins are produced by overnight bacterial cultivation, before the more fun (and stressful) experimental work could begin. On the first day of protein production, the bacterial cultivation was started, followed by induction by IPTG. On the third day we could finally start the purification. This included many steps, like sonication, a bunch of centrifugation, and affinity chromatography as purification method. We used SDS-PAGE to check the success of the protein production and purification. The purification and analysis done afterwards usually took a whole day, with addition of a couple more hours if we started concentrating the protein.

Hopefully after this we would have a good amount of protein on which the assays could be performed during the rest of the week. How did it go then? Well, at first things did go according to this schedule, but as with many things in life, they do not always go exactly as planned. It is common when testing the production yield and stability of new enzymes that several of the production steps need to be repeated or optimized to get them perfect. In some cases, if it got really late to be in the lab, we could postpone one task until later the same day or the next day, but in other cases (like when you mix the proteins up or have contaminated growth media) we had to start again from the beginning. 

When we started as research interns, we didn’t imagine that the greatest thing that the internship would teach us would not be laboratory skills but self-confidence and patience. There were many moments when we asked ourselves “What else could possibly go wrong!?” We definitely sometimes felt disappointed and disheartened maybe due to some failed experiment. What we learned from this was to own our mistakes, learn from them, and move forward without dwelling on the failures too much. In these times it was very helpful to have our supervisors there to guide us with their knowledge. These frustrating and unexpected experiences really shaped us and helped us learn from them and grow personally.

Our final message: it’s okay to shed some exasperated tears. In the lab, we know we can expect to face many moments where everything seems to be going wrong, and that we’ll feel every emotion that comes along with that. But then, we lean on our colleagues, we learn from our experiences, and we do everything possible to overcome the challenges before us. Everything may not go as planned, but we do everything in our power to make the outcome golden. Lastly, we would like to thank Lauren, Mengshu, Heli, and the CAZyme team for giving us this great opportunity and experience that will last a lifetime.

Reflections – Project work in the CAZyme group🌟

August 2021 has been my eighth month working with Lauren and the CAZyme group. It would be a lie if I say there has not been one time that I felt exhausted – what other kind of mood should one have when a just-autoclaved waste bag that is full of agar plates is leaking!? However, I genuinely enjoy every day, and every bit of progress that we have reached here in the biocontrol project.

At the very beginning of my time with the group, I was a master thesis student. From several interesting lectures Lauren gave in my program, Industrial and Environmental Biotechnology (starting Autumn ’19) at KTH, I was hugely intrigued by environmental biotechnology and its applications incorporating sustainability, such as agri-biotech, green food-tech, etc. The importance of microbiology in dealing with environmental contamination is another big aspect that cannot be overlooked. Among the various kinds of microbial application in environmental science and engineering, I have an interest in green agriculture, where the well-known downfall of large-scale commercialized production of crops has endangered the environment more and more drastically via over-fertilization, mono-species cultivation, over-irrigation, etc. Many of these issues have causal links to disproportionate chemical usage, and the abuse of pesticides as well as fungicides is another prime culprit.

Everyday our crop plants are fighting against pathogenic attack. For us to help them survive and grow, the most commonly used measure in this fight is for farmers and growers to apply chemicals to repel the invasion of pathogens. While this might lead to powerful short-term positive results, the tragic side effect lies within the soil, where the supposedly healthy microbial community is destroyed along with the pathogens, if the chemicals aren’t selective. The unbalanced soil consortium is then slowly losing its resilience to recover from the damage caused either by pathogens or chemicals. This is of course ironic, because the chemicals were supposed to be helping the plants!

The theme in green agriculture that I have investigated throughout my project is about “Biocontrol”. I work with a particular soil-inhabiting bacterium, C. pinensis, which is likely to interfere in the ability of pathogens to attack plants, thanks to its own pathogen cell wall-degrading potential. I try to amplify the growth of the bacterium and further mediate its secretion of pathogen-degrading enzymes. The enzymes secreted might be the key to unlocking an alternative to commercial fungicides.

Thanks to other master students and preliminary studies done in the CAZyme lab, I continued the C. pinensis project: I was in charge of testing for in vitro inhibition performance of the bacteria-derived enzymes against pathogenic oomycetes. The oomycetes chosen are ones that can attack plants and bring about severe crop diseases, for instance, root rot, blight, etc. I hope that in the near future you’ll be able to read a publication showcasing my data, but in short, I managed to boost bacterial enzyme secretion through supplementing different kinds of carbon sources in bacterial cultures. Up to now, combined with results from other team members, our hypothesis leads to quite a positive vindication – the carbon source provided and the enzymes henceforth secreted are certainly affecting the bacterium’s inhibition ability in a better than expected way. I won’t give you too much more detail for now 😉

To be able to play a part in this agriculture-related biotechnological research is a great pleasure of mine, for I am concerned with food justice, and to solve major agricultural issues with more sustainable methods is definitely an ultimate goal in the long-term food production scheme: A more recovered soil status means a more fertile/healthy growth condition for crops, which will eventually lead to a better yield in the hope of easing hunger problems. Of course, the issue of hunger is complex to deal with, and only one step forwards towards sustainable production is not enough. But why not see this as a positive step, showing we are heading somewhere, somewhere food justice is to be fulfilled little by little?

Though having had project experience back in my bachelor years, to conduct a research thesis project was an entirely new experience for me, let alone to have the opportunity to extend the project into a paid summer internship, significantly advancing my work through the summer following my graduation. I feel grateful for this chance, and for the advice I have gained from Lauren and all who have helped me in this journey.

Life in the lab feels a bit like working around the train schedule; there exist peak hours and off-peak hours. In the peak hours where several people are waiting to use the same equipment, we learn how to plan ahead better and how to find common benefits via efficient communication; during the off-peak hours, like when the precious summer sun comes out and says “Hi!”, we have the chance to calmly spend time in whatever our work is, listening to ourselves and appreciating the privilege of having access to knowledge. This is what higher education and a scientific career is for, I suppose, to repay what we have received from our teachers, by heading elsewhere to make new efforts in research. The train never stops.

The project is currently still going onwards with many endeavors. Thanks sincerely to Lauren and the CAZyme Group for welcoming and guiding me when executing the project. Even when I was sometimes unsure of which direction to follow in the project, the strong communication and positive working dynamics have never failed to give firm hands, gently assisting step after step. I am glad that I have contributed to some of the results. Let’s finger-cross for cheerful updates on the biocontrol project in the near future!