Introduction

Our project mainly consisted of 2 experimental parts:

• Finding the best SCFA
• Increasing the concentration of this SCFA through a probiotic

We will describe below some basic information about our experiments and our results, the exact protocols we used can be found in the Lab Book.

How did we choose to work with butyric acid?

After our bibliographical research, we found out that the two most important SCFAs for the gut-brain communication are acetate and butyrate.

Read more about the SCFAs in our Background Knowledge.

In order to identify which one of the two fatty acids would be the best fit for the needs of our own project, we performed our first experiment. As we couldn’t use animal models, we decided to make the compromise to use an enteric cell line, believing that this would provide a simulation of the gut. More specifically, our first experiment was based on the ability of some enteric cells to produce serotonin, as a response to the existence of SCFAs in their environment. This way, we thought that we could have some first data showing that the SCFAs increase the serotonin production (and as a result alleviate the depressive symptoms, as explained in the Project Description).

Initially, we planned to use the R1N14B cell line, a cell line exhibiting epithelial morphology that was isolated from the pancreas of a male rat. R1N14B cells are enterochromaffin cells that synthesise ∼95% of the body's total serotonin (5-HT). However, as our lab’s faculty was trained to use the Caco-2 cell line, after studying the corresponding bibliography and research recent papers, we adapted our project into using the Caco-2 cell line.

After successfully culturing a Caco-2 cell line, we added the two SCFAs in the following concentrations:

First Component	Second Component
Acetate Low (AL): 10mM	Acetate Medium (AM): 20mM
Acetate High (AH): 30mM	-
Butyrate Low (BL): 0,5mM	Butyrate Medium (BM): 1mM
Butyrate High (BH): 1,5mM	Butyrate Extreme (BE): 2,5mM
ALBL: 10mM + 0,5mM	ALBM: 10mM + 1mM
ALBH: 10mM + 1,5mM	-
AMBL: 20mM + 0,5mM	AMBM: 20mM + 1mM
AMBH: 20mM + 1,5mM	-
AHBL: 30mM + 0,5mM	AHBM: 30mM + 1mM
AHBH: 30mM +1,5mM	-

Our goal was to investigate whether butyrate, acetate, or their combination would be the most efficient for serotonin production. We measured the serotonin produced using a serotonin ELISA Kit (https://www.immusmol.com/shop/serotonin-elisa-kit-any-sample/). We ran the same experiment twice. However, the results coming only from the second experiment are presented below. In the first experiment, we used extremely high concentrations of SCFAs, and, as a result, the kit didn’t work properly.

Results:

The analysis of our results indicated that among the two primary SCFAs we examined, acetate appears to play a slightly more significant role in influencing final serotonin levels. Notably, the maximum serotonin concentration is achieved when both acetate and butyrate are present at their highest concentrations, as shown in the plot below. More about the methods we used to analyze our results can be found in the Model.

From the barplot above we can observe that:

• Butyrate when administered with no acetate, causes less serotonin production than the control (and than acetate administrations).
• For a standard butyrate concentration, increasing acetate tends to increase serotonin production. In some cases (B00 and B10) , a decrease is observed from A20 to A30.
• The most efficient combination on serotonin production is B10_A20
• The B15_A10 combination had almost the same serotonin production as the control.

How do we plan to increase the SCFAs’ concentration?

As mentioned above, we found that the maximum serotonin concentration is achieved when both acetate and butyrate are present at their highest concentrations.

We chose to work with Lactobacillus rhamnosus gg, a commonly used probiotic. Lactobacillus rhamnosus gg normally produces acetate. So, our team’s idea was that, if we could manage to make this strain produce butyrate as well, we would achieve the maximum serotonin production when administering the probiotic. In order to achieve this, we genetically modified Lactobacillus rhamnosus gg, by inserting a plasmid containing the necessary genes.

This part of our experiments took place in the Laboratory of Biotechnology and Applied Microbiology.

We intended to run two separate experiments for the genetic modification of the Lactobacillus strain. In the first experiment, we would transform the cells using the old iGEM part (BBa_K1618021), while in the second we would use our own new improved part (BBa_K4989008). In this section there is no information about the parts, but you can read more about them in the New Improved Part section.

Although Lactobacillus rhamnosus gg is our probiotic strain, we also used E.Coli K12 to better characterize our parts.

We started off by measuring the concentration of the SCFAs normally produced by the two strains in a 24h culture, to use these measurements as a control. These values were determined by HPLC, using a Shimadzu chromatography system.

Afterwards, we proceeded to transform both of the bacteria strains. We cloned our insert in the pNZ8149 food-graded plasmid, using Golden Gate methods and enzymes. We also used IPTG to induce the expression of the old part, as it was controlled by a lac-promoter. After succesfully transforming both E.Coli K12 and L.rhamnosus gg with the plasmid containing the old part (conducted colony PCR to check the transformation) and cultured them for 24h, we measured their production of SCFAs as well, using HPLC.

Sadly, we faced many difficulties ordering the new part. We ordered it from two different suppliers multiple times, but we experienced many delays because of its length and complexity. As a result, the new part didn’t arrive on time and we are still working on this experiment. Although we won’t be able to have these results by the Freeze Day to upload them on our Wiki, we will continue our experiments and we will for sure share our updates with the judges in the judge Q&A during the Grand Jamboree!

We understand that our New Improved Part insert is considerably long, which is one of our major concerns. However, we have very carefully designed every part of our insert, so we are optimistic that it should work and we expect to observe an increase in the production of butyrate. In order to identify whether the strains have eventually been transformed with the plasmid, we have included a GFP protein sequence to our part.

Results:

We can see that before the transformation, as expected, there is production of acetate and lactate. E.Coli also produces a very low quantity of butyrate.

We can observe that there is no big difference when inserting the old part. This aligns with previous teams’ research, as the part didn’t work for them as well.

As we mentioned above, we still don’t have the results from the transformation with the plasmid containing our new improved part. However, what we are expecting to observe is green fluorescent cultures, producing high concentrations of lactate and acetate , along with a higher concentration of butyrate. We will be happy to discuss with the judges our results in the judge Q&A during the Grand Jamboree!