Human Practice

At iGEM Erlangen, we firmly believe that meaningful scientific research extends beyond the lab. It involves understanding the human aspects that surround our project and actively seeking insights from experts in the field. Our human practice efforts took us on an enlightening journey, including a meeting with Prof. Zaiss. Here, we share the highlights of our interaction:

Unleashing Innovative Ideas

Our conversation with Prof. Zaiss was a hub of creative brainstorming. We contemplated several groundbreaking ideas related to our project, including:

Detecting SCFAs: We explored the notion of engineering bacteria to emit green fluorescence when they produce short-chain fatty acids (SCFAs). This could serve as a fascinating indicator of an individual's microbiota competence, revealing their ability to produce SCFAs based on their diet and microbiota. We discussed the challenges of working with anaerobic bacteria and the potential adaptability of this protocol to different bacterial strains.

OMV Modification: We considered the intriguing prospect of using outer membrane vesicles (OMVs) to express beneficial receptors for SCFAs in the intestine. However, we acknowledged that this approach posed technical challenges.

Enhancing SCFA Production: Our minds wandered to the idea of creating bacteria that could produce an abundance of SCFAs. This would involve overexpressing enzymes responsible for generating protective SCFAs while inhibiting those responsible for inflammatory SCFAs. Although this approach could potentially offer a way to alter SCFAs through dietary changes, it was deemed technically demanding.

Investigating SCFAs: We pondered the possibility of conducting experiments to measure the impact of different SCFAs on various cellular parameters, such as inflammatory cytokines and gut integrity. We even considered utilizing a "gut-on-chip" device to replicate the intestinal environment.

Mimicking SCFAs: Exploring the creation of SCFA agonists captured our attention. These agonists could provide an alternative to dietary changes, potentially involving collaboration with chemists or dry lab techniques to study how SCFA activity changes with these mimics.

Antibody-Based Approach: Another intriguing concept was the development of antibodies that activate SCFA receptors. This could offer assistance to individuals who struggle to consume a diet rich in SCFAs, such as celiac patients. These antibodies could be used alongside detection methods to identify cases where individuals lack sufficient SCFAs.

Putting Ideas into Action:

Prof. Zaiss emphasized the importance of initiating experiments that are not only innovative but also feasible. He encouraged us to establish a clear framework for each idea and suggested practical steps, such as growing E. coli with modified pathways to emit green fluorescence and exploring the possibility of expressing receptors on bacteria.

General Takeaways:

Our conversation with Prof. Zaiss underscored the significance of setting milestones to demonstrate the viability of our ideas. He highlighted the importance of effectively communicating why our project matters and the value of crafting a compelling presentation. Ultimately, Prof. Zaiss stressed that having a strong, innovative idea is more crucial than achieving a flawless outcome.

A Dry Lab delegation met with Prof. Burkovski at Department of Biology to talk about metabolic modelling and how a Dry Lab approach could aid our project most.

Important things to keep in mind when modelling bacteria

procaryotes are much more flexible when it comes to adapting their biochemical composition to environmental changes. By switching between sigmulons bacteria can quickly shift their protein production to a completely different mode of action. On the one hand, this makes it difficult to predict how E.coli will act in certain situations since a proteomic shift, due to stress factors or different available energy sources, might fundamentally change it's properties. On the other hand this issue makes it difficult to modify the metabolism of E.coli so that for example acetate would not be turned over any more. Prof. Burkovski suggested us to first find a preferably simple operon controlling the SCFA metabolism as a starting point for further plans. Since higher levels of regulation like regulons or modulons possess usually a lot of cross effects in bacteria, it would be easiest to just work with operons to achive our goal.

As a general advice for us as a Dry Lab team, Prof. Burkovski recommended us to develop a back-up plan for our current wet lab approach. We could also calculate and research options for effectively storing and testing the transformed bacteria. A central question we should think about was which energy source would be offered to the bacteria while they were exposed to samples of SCFAs.

We want to thank Prof. Burkovski cordially for supplying us with a lot of background information and expertise about E.coli. His recommendations were vital for our later Dry Lab projects.

A part of our Dry Lab team met with Prof. Uwe Sonnewald at the Chair of Biochemistry of the FAU. We presented our plan on using a metabolic model of the endogenous acetate pathway in E.coli to simulate it’s interaction with our biosensor construct. We asked for Prof. Sonnewalds opinion on our idea and also the project in general.

In summary, we discussed the following points:

Endogenous Metabolism & Sensitivity

The main obstacle we are facing will probably be the sensitivity of our sensor system. If a lot of acetate turn-over takes place inside the cells, it will be difficult to detect the signal derived from the acetate in the medical sample. Therefore, we should aim to establish conditions which induce as little acetate catabolism as possible. This could for example be achieved by supplying E.coli with an appropriate carbon source. The best option would be to suppress the operons catabolising acetate while keeping the membrane transporters for acetate active.

2^nd Reporter & Calibration

To make calibration easier, Prof. Sonnewald suggested to express a second reporter constitutively in all our transformed cells. If the signal from this reporter changes after adding the bacteria to the sample of faeces, we could conclude that the cell count or over all expression rates changed due to the altered conditions. If this influence cannot be eliminated, at least the second reporter supports mathematical normalization of the desired signal indicating the SCFAs.

While taking measurements with a FACS device, an 2^nd reporter will not be obligatory. But for the final detection kit it might provide independence from access to FACS.

Modelling & Mass Spectrometry

A problem with metabolic modelling will be the rapid adaption of procaryotic enzyme concentrations to changing environment conditions. It cannot be assumed, that kinetic parameters of intracellular reactions remain constant over extended periods of time, especially during the change of outside conditions. The same issue applies to intracellular metabolite concentrations under the conditions we are going to work with. To tackle this, Prof. Sonnewald offered us to analyse E.coli cell extracts in the mass spectrometer of his chair.

This could already be done before the transformation of our bacteria is complete, just to get the right metabolic data for our model. After successful transformation, this friendly offer can also be very useful for calibrating our system.

We cordially thank Prof. Sonnewald for sharing his knowledge with us and for offering his support in form of mass spectrometry measurements.

To broaden our knowledge about the role of SCFAs in the body, our integrated human practice team conducted an expert interview with our supervisor Prof. Dr. Mario Zaiss.

Prof. Dr. Zaiss works at the Institute for Clinical Immunology at the University of Erlangen-Nürnberg, Germany, where he researches the effect of the microbiome in rheumatoid arthritis (RA) and how these new insights can be used to treat rheumatoid arthritis patients.

First, Prof. Dr. Zaiss told us more about the microbiome in relation to rheumatoid arthritis: patients affected by the disease have been observed to have microbial dysbiosis, a reduction in the diversity of beneficial gut bacteria. Additionally, others who are at high risk of developing rheumatoid arthritis were also seen to have this disruption in the microbiome. This supports the idea that microbial dysbiosis is an environmental trigger for RA.

Moreover, we learned about the impact diet has on the natural balance (homeostasis) of the bacteria within the intestines. One important element of the diet is fibre, which can be soluble or insoluble. Insoluble fibre aids the movement of solids through the intestines, whereas soluble fibre is absorbed by certain gut bacteria and fermented to produce metabolites. Among the most abundant metabolites are short-chain fatty acids (SCFA) which serve as a source of fuel for intestinal epithelial cells and other bacteria types, consequently regulating the processes within the gut.

Besides that, we were also able to discuss a part of Prof. Dr. Zaiss’ research, that focuses on the impact of a high fibre diet on RA patients, with him. For this purpose, different immune parameters present in blood and stool samples of rheumatoid arthritis patients were monitored after the introduction of a high fibre diet. It became evident, that an increase of fibre in the diet, led to an increase of circulating regulatory T cells, which work to suppress the immune system to prevent autoimmunity and over-inflammation. Excitingly, a decrease in markers of bone erosion in affected joints, could also be observed. Therefore, it would be a possibility to supplement or complement existing pharmacological treatment approaches for RA. In addition to their anti-inflammatory properties, it was also found that SCFAs promote regulatory T cells to carry out their function and some evidence suggests that they regulate other inflammatory immune molecules like chemokines.

After learning so much about SCFAs, the microbiome and Prof. Dr. Zaiss’ research, we wanted to know more about other potential roles SCFAs play in the body. Here, we learned that alcohol consumption can protect the body from autoimmune diseases like RA. This could be explained by Prof. Dr. Zaiss and his colleagues, who found out that alcohol metabolizes into acetate in the body. Acetate alters the functionality of T helper cells which aids to regulate the immune system and prevent inflammation. This effect of acetate on the T helper cells means that the production of autoantibodies (antibodies aimed against the host’s body) is inhibited. Consequently, the autoimmune impacts of rheumatoid arthritis are reduced.

Lastly, Prof. Dr. Zaiss introduced us to the mechanisms of gut microbial dysbiosis that cause rheumatoid arthritis. We learned that a peptide called zonulin is found in unusually high quantities in autoimmune humans and mice. Zonulin has a vital part in regulating the tight junctions between cells that prevent water and its solutes from leaking where they shouldn’t. When too much zonulin is present in gut cells, the intestinal barrier becomes leaky and dysbiosis and inflammation occur. Therefore, high blood levels of zonulin can be used to predict the onset of clinical arthritis from asymptomatic autoimmunity. Additionally, it was found that certain compounds such as the SCFA butyrate can prevent RA, by restoring the intestinal barrier before arthritis can take hold. In addition, larazotide acetate, which works to prevent the action of zonulin, increases the integrity of the intestinal barrier to reduce the onset of arthritis. These insights could be used in the future to develop drugs that minimise intestinal barrier damage in order to stop clinical rheumatoid arthritis from forming.

All in all we thank Prof. Dr. Mario Zaiss for this great interview opportunity. Our interview further showed us the important role SCFAs and the microbiome play in the body.