iGEM 2023 — Team fau-erlangen

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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## January

* ### 12.01.2023

  Team Meeting.
* ### 28.01.2023

  Dry Lab:

  First Meeting, discussing about different methods, aptamers, SCFAs and open sources for proteins.

## February

* ### 01.02.2023

  Wet Lab:

  General brainstorming about possible project ideas to discuss with Prof. Zaiss.
* ### 03.02.2023

  Wet Lab:

  Meeting with Prof. Zaiss and discussing the ideas we came up with.
* ### 07.02.2023

  Finances:

  First Meeting to discuss possible financing opportunities.

  Human Practices:

  Brainstorming about general project ideas.
* ### 08.02.2023

  Wet Lab:

  Sorting through and categorize our ideas, rough first workflow.
* ### 09.02.2023

  Dry Lab:

  First Discussion with Prof. Böckmann + new plan to use open source microbiome data and make comparison with wetlab results.
* ### 15.02.2023

  Wet Lab:

  Searching for SCFAs receptors and experiments we could perform to detect their activity.
* ### 18.02.2023

  Human Practices:

  Preparation of questions for the general public Pol and future Interviews + planning the podcast.
* ### 20.02.2023

  Human Practices:

  Writing the first podcast script.
* ### 22.02.2023

  Wet Lab:

  Collecting ideas to detect SCFAs: quantification with split GFP-assay or measure claudin unregulation or measure FFAR2 phosphorylation.
* ### 25.02.2023

  Wet Lab:

  New idea: Microfluidics device to detect SCFAs via binding to respective enzymes.

## March

* ### 01.03.2023

  Wet Lab:

  Discuss research about microfluidics, biosensors, enzymes, plasmids.
* ### 03.03.2023

  Human Practices:

  Deciding on initial pool of questions for the general public Pol.

  Dry Lab:

  Generating ideas, introducing the Human Microbiome project, research on IBD sources.
* ### 08.03.2023

  Dry Lab:

  Sharing individual research, collecting ideas, deciding on a python schedule.
* ### 10.03.2023

  Human Practices:

  Making the introduction sound for the podcast series.
* ### 16.03.2023

  Finances:

  Starting the first draft of the grand application.
* ### 22.03.2023

  Wet Lab:

  Discuss suitable enzymes, fluorochromes and optical biosensors.

## April

* ### 05.04.2023

  Finances:

  Distributing the different sections of the grand application.

  Wet Lab:

  Further research and discussing about suitable enzymes, on how to detect fluorescence and about plasmids for bacterial transformation.
* ### 12.04.2023

  Wet Lab:

  Discussing and choosing operons for SCFA detection, selecting fluorochromes with no/little spectral overlap and considering which plasmid would fit our purpose.
* ### 19.04.2023

  Wet Lab / Finance:

  Create presentation for biology department
* ### 20.04.2023

  Finances:

  Presenting our topic and iGEM to the Biology Department.
* ### 23.04.2023

  Human Practices:

  Translation of the general public Pol into German, Spanish and Turkish.

  Wet Lab / Finance

  Create presentation for chemistry department.
* ### 24.04.2023

  Finances:

  Presenting our topic and iGEM to the Chemistry Department.
* ### 25.04.2023

  Dry Lab:

  Meeting with Prof. Burkovski: Modelling part abandoned, omparison between the genome of two species to identify similar genes.
* ### 26.04.2023

  Meeting of Dry Lab Team with Wet Lab team to discuss how dry lab could integrate wetlab results.

  Human Practices:

  Release date of the general public Pol.

  Brainstorming about potential collaboration ideas.

  Preparation of a list with potential schools and games for public outreach/education.

## May

* ### 02.05.2023

  Human Practices:

  Recording and editing of the first podcast episode.
* ### 03.05.2023

  Wet Lab:

  Gathering sequences for promotors and fluorochromes, sort experiments we will conduct and further plan plasmid design.
* ### 04.05.2023

  Wet Lab:

  Meeting with Prof. Burkovski to clarify questions about plasmid.
* ### 06.05.2023

  Dry Lab:

  Collecting ideas: Calculators, games, etc. + Distribution of research about specific genes.
* ### 10.05.2023

  Human Practices:

  Designing and colouring games for the schools.
* ### 16.05.2023

  Finances:

  Getting financial support for laboratory materials from Pandora.

  Dry Lab:

  Potential gene candidates discussed.
* ### 17.05.2023

  Human Practices:

  Writing scripts for podcast episodes.

  Establishing podcast subteams.
* ### 20.05.2023

  Human Practices:

  Recording of podcast episodes.

  Dry Lab:

  Preparing questions for meeting with Prof. Burkovski.
* ### 21.05.2023

  Fiances:

  Proof-Reading the grand application and starting to send it to various foundations/companies.
* ### 24.05.2023

  Wet Lab:

  Task distribution: start designing our constructs i Benchling, find experiment protocols, write a list what materials we will need in the lab.
* ### 31.05.2023

  Fiances:

  Sending away our application for the Team Impact Grand.

  Wet Lab:

  Create rough time plan for the lab work, ask Prof. Koch and Prof. Sonnewald for glnAp2 sequence and ask Prof. Winkler to provide E.coli.

## June

* ### 03.06.2023

  Human Practices:

  Preparing new scripts, recording and editing of the trailer for the podcast.
* ### 06.06.2023

  Human Practices:

  Brainstorming about potential meetings/interviews with patients, experts and companies.
* ### 07.06.2023

  Wet Lab:

  Discuss cloning methods and tags to save primer for PCR.
* ### 10.06.2023

  Human Practices:

  Recording and editing of podcast episodes.
* ### 14.06.2023

  Wet Lab:

  Create experiment schedule to plan experiments and double check our protocols.
* ### 16.06.2023

  Dry Lab:

  Meeting with Prof. Burkovski.
* ### 27.06.2023

  Human Practices:

  Contacting schools and pharmacological companies.

## July

* ### 05.07.2023

  Wet Lab:

  Introductio of the wet lab team to Prof. Slany, Prof. Nimmerjahn, Prof. Lux and TA, finalize Benchling constructs so IDT sequence can be ordered.
* ### 08.07.2023

  Human Practices:

  Recording and editing the podcast.
* ### 11.07.2023

  Dry Lab:

  Alternative idea: kegg pathway analysis + further research about kegg pathway.
* ### 12.07.2023

  Wet Lab:

  Preparing LB agar plates.

  Discussing next steps and what can be prepared while IDT sequences are ordered.
* ### 18.07.2023

  Human Practices:

  Preparing a questionnaire for IBD patients + Interview script to talk with Prof. Zaiss.
* ### 20.07.2023

  Dry Lab:

  Designing the primer calculator.

## August

* ### 08.08.2023

  Dry Lab:

  Contacting Prof. Sonnewald for scientific advice related to kegg pathway.
* ### 09.08.2023

  Wet Lab:

  Meeting with Prof. Winkler to discuss our procedure in the lab.
* ### 10.08.2023

  Human Practices:

  Meeting with Prof. Sonnewald.
* ### 14.08.2023

  Dry Lab:

  Finishing and Documenting the primer calculator.

  Wet Lab:

  IDT DNA elution and first amplifying PCR.
* ### 15.08.2023

  Human Practices:

  Outlines for the interview with Prof. Zaiss and Prof. Atreya.

  Dry Lab:

  Meeting with Prof. Sonnewald.
* ### 16.08.2023

  Wet Labs:

  Gel electrophoresis to confirm PCR.
* ### 23.08.2023

  Wet Lab:

  First Gibson Assembly and bacteria transformation.
* ### 27.08.2023

  Human Practices:

  Recording a podcast episode.

## September

* ### 09.09.2023

  Human Practices:

  Recording + editing the next podcast episode.

  Translating the IBD patient questionnaire to English and German.
* ### 12.09.2023

  Meeting of Dry Lab and Wet Lab about samples for mass spectrometry + Model preparation.
* ### 16.09.2023

  FAUgem trailer is aired.
* ### 17.09.2023

  Human Practices:

  Recording podcast episodes.
* ### 22.09.2023

  FAUgem S1E1 is aired.
* ### 26.09.2023

  Dry Lab:

  Finalizing the model.
* ### 30.09.2023

  FAUgem S1E2 is aired.

  Human Practices:

  Editing podcast episodes.

## October

* ### 03.09.2023

  Dry Lab:

  Finalized Model prepared and shared with the team.

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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## Construct Engineering

To be able to use Gibbson Assembly we needed to add overlapping overhangs to our construct. The first time we designed them, we chose XbaI as restriction enzyme and primers that would match our constructs and the restriction site. We then faced the problem, that the G-C content was too high to ensure good PCR results. To overcome this issue we designed a two-step PCR for the primers for acetate, butyrate and prpR: In the first step we used primers that added sequences that are low in G-C to the primers for our construct and therefore enabled another set of primers in a secondary PCR to then bind to those sequences. After trying different temperatures and primer concentrations, we still had troubles doing the first step PCR, indicating that the G-C content was already too high for the first set of primers to bind. We solved the problem by choosing another restriction site based on a low G-C content at the neighbouring bases and used the restriction enzyme Hind3. Because of the change we could directly design primer that added the needed overhangs in one step.

## Endogenous Acetate Knockout

Since bacteria also produce endogenous acetate, we were afraid that this would influence our measurements of external acetate. One idea we had, was to produce an acetate knockout. We discussed our ideas with Prof. Winkler, who told us that we could use the CRISPR-Cas9 system for that, but that we would need to be careful to ensure those modified bacteria would still need to be able to live without the acetate. We then thought about adding acetate to the media and remove it before starting the tests. In the future we would then need to measure, how long those bacteria can survive without endogenous acetate and when adding it to the media, how we would need to deprive our bacteria before starting the test series.

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* [Results](#results)
* [Discussion](#discussion)

## Results

### Bacterial Transformation

---

To transform NEB-5alpha competent bacteria with our reporter constructs and create our GLOW.coli, we agreed to perform a Gibson Assembly. To do this, we started by thinking about the best way to order our inserts - consisting of an inducible promoter on the one hand and a reporter gene (GFP, BFP, TaqRFP) on the other. To do this, we used the Benchling platform, which enabled us to download all the sequences we needed and carry out a Gibson assembly virtually. We agreed to linearise our plasmid pBluescript KS (+) with the enzyme HindIII, as it only occurs once in the plasmid and also has an optimal GC ratio after inclusion of the inserts, which should alleviate PCR. We took advantage of the fact that Gibson assembly requires overhangs that are complementary to the vector by inserting HindIII restriction sites once forward and once reverse into the extensions of the sequences. These do not interfere with the coding sequences but allow the insert to be accurately excised from the plasmid, provided it has been correctly inserted into the plasmid. Accordingly, we would expect to see bands as shown in Figure 1. You can see a virtual digest with HindIII, for Acetate, Butyrates and Propionates for all three reporter genes. The plasmid is 3007bp long, so a band at the appropriate level is expected. The acetate promoter glnAP2 (150 bp) together with the reporter gene GFP has a length of 976 bp, with BFP a total length of 979 bp and with TaqRFP a length of bp. Correspondingly, the bands for the butyrate promoter (pchA: 82bp) appear in the virtual digest. Since the propionate construct contains an additional regulatory sequence (prpR: 1587 bp) as an additional insert, which creates an additional HindIII restriction site in the insert, two additional bands should appear after a successful digest, in addition to the plasmid (promotor prpB: 62bp).

![HindIII vitrual digest](https://static.igem.wiki/teams/4930/wiki/results/virtaul-digest.png)

Fig.1: HindIII vitrual digest

### Gibson assembly

---

The first step of our cloning process involved the linearization of our plasmid with HindIII. To confirm successful digestion, we performed an agarose gel electrophoresis. In addition to the undigested vector as a control, we ran two samples of digested plasmid (c1 and c2). These two samples only differed in the volume of the enzyme used (c1 1µl vs. c2 1.5µl), with no noticeable difference observed. In both cases, we were able to detect linearized plasmid in the gel. Unlike the uncut vector, the linearized plasmid migrates more slowly through the gel due to its open linear structure, which can bind to the gel matrix and therefore move more slowly compared to the undigested, circular, and compact ring-shaped structure. As can be seen from Figure 2, the linearization was successful, so we could use the vector for the Gibson assembly

![Vector linearization](https://static.igem.wiki/teams/4930/wiki/results/vektor-lin.png)

Fig.2: Vector linearization

After performing the Gibson assembly according to the protocol (refer to [Lab Book](https://2023.igem.wiki/fau-erlangen/labbook) under Gibson Assembly), transforming the bacteria, and streaking them on LB agar plates, we first checked the negative control plate the following day. As expected, the negative control plate showed no colonies, in contrast to the ones with the transformed bacteria. This prompted us to decide to pick colonies to inoculate fresh LB medium.

After eight hours of growth, we used 3 mL oh the suspensions for DNA purification performin a Mini-Prep and 0.5µl of bacterial suspension for a Colony-PCR. The purpose of both methods was to verify the presence of our insert.
As mentioned above, we incorporated HindIII sites designed to facilitate the removal of inserts at specific locations. Consequently, after DNA isolation, we performed a digestion with HindIII and subsequently loaded the DNA onto a 2% agarose gel. Upon examining Figure 3, one can observe bands that are smaller than the uncut vector and bands that are larger than the uncut vector (labeled as Plasmid). The higher band served as an indication to us that the enzyme worked. As the 1kb also could not be resolved even after an extended period, we mistakenly assumed the smallest band to be the insert. We attributed the DNA getting stuck in the well and slowed DNA migration through the gel to an excessively high gel percentage.

![Restriction digestion to confirm Gibson assembly](https://static.igem.wiki/teams/4930/wiki/results/digest.png)

Fig.3: Restriction digestion to confirm Gibson assembly

Figure 4A shows the Colony-PCR results for the Acetate constructs. 'Pro' stands for the primers that only amplify the promoter, while 'Rep' represents the primers that amplify the respective reporter genes. Using the same principle, Figures 4B and 4C were generated for Butyrate and Propionate. Figure 4D shows an attempt to amplify the regulatory sequence prpR. It can be stated that in none of the figures, the Colony-PCR was successful. Bands are visible, but these correspond to non-specific amplifications. Accordingly, we concluded that the Gibson Assembly did not work. Subsequently, by performing a Primer-Blast, primer binding sites in the genome of the used E. coli strain were identified. Since the generation of the primers was based on plasmid DNA and not genomic DNA, this binding was unforeseen. One additional reason the Gibson assembly didn't work is that in the process of vector linearization, we used alkaline phosphatase to remove the phosphate ends to prevent re-ligation. However, we did not consider that this process would interfere with the Gibson assembly.

![First Colony PCR](https://static.igem.wiki/teams/4930/wiki/results/colony-pcr-11.png)

Fig.4: First Colony PCR

By using the forward and reverse primers, which, unlike the other primers, do not have primer binding sites within the inserts, we were able to detect the plasmid at the correct size of about 3007 bp for all constructs (Figure 5). This suggests the functionality of the primers.

![PCR to confimr Plasmid length](https://static.igem.wiki/teams/4930/wiki/results/pcr-plasmid.png)

Fig.5: PCR to confirm Vector length

Since all the results indicated that the Gibson Assembly did not work and we suspected dephosphorylation as the cause, we proceeded to conduct another Gibson Assembly, this time without dephosphorylation. We followed the Gibson protocol as before and used the same plasmid. Prior to streaking the transformed bacteria on the plates, we spread X-Gal (5-Bromo-4-chloro-3-indolyl-β-D-galactoside) on the LB agar plates. This allowed us to perform a blue-white selection. For blue-white selection, a plasmid can be used that contains the gene for β-galactosidase (lacZ gene) at the position where the transgene is inserted into the plasmid (Multiple Cloning Site). Inserting an insert into the Multiple Cloning Site inactivates the galactosidase. As a result, after transformation, the transgenic organisms, in contrast to the non-transgenic organisms, do not contain a functional galactosidase, causing their colonies to remain white, allowing to be isolated based on their lack of color (Fig. 5).

![Confirmation of second Gibson assembly via blue-white selection](https://static.igem.wiki/teams/4930/wiki/results/xgal-digestion.jpg)

Fig.6: Confirmation of second Gibson assembly via blue-white selection

As seen in Figure 6, both white and blue colonies grew. So we used a pipette tip to pick the white colonies and subsequently conducted another Colony-PCR. As Figure 7A demonstrates, bands in the Propionate constructs are visible and correspond to the expected band lenghts. The labelling corresponds to Figure 4.

![Second colony PCR](https://static.igem.wiki/teams/4930/wiki/results/second-colony-pcr1.png)

Fig.7: Second Colony PCR

The insert containing the Propionate promoter includes an additional spacer to extend the short promoter with 62 base pairs to 252 base pairs. In the GFP and BFP constructs, a band at approximately 250bp can be detected, which supports the amplification of the correct band. Additionally, in the wells containing the primers for the reporter genes, bands with 750 base pairs can be observed. Since the reporter genes, GFP, BFP, and TaqRFP, all range between 740 and 750bp, this band also indicates a successful amplification of the desired insert.

Figure 7B displays the Colony-PCR using primers for the regulatory sequence prpR, which is 1616bp long. A prominent band is visible in the RFP construct, which does not exist in the other constructs (exceeding 5000bp). As the entire construct, including the promoter and regulatory sequence, is 5702bp in length, the amplification of the entire construct cannot be ruled out. Overall, it can be concluded that the Colony-PCR for Propionate suggests that the Gibson Assembly worked. Due to a lack of time, planned experiments with the successful Gibson were not conducted in a timely manner but will be continued in the future.

### GLOW.coli

Despite the fact that our Gibson Assembly did not work, we proceeded to use our constructs and incubated them with Short-Chain Fatty Acids (SCFAs) as outlined in the lab notebook. Keeping our failed Gibson Assembly in mind, our aim was nevertheless to investigate whether any difference in fluorescence could be observed when exposing the same number of bacteria to different concentrations of SCFAs. To accomplish this, we utilized all nine constructs (Acetate, Butyrate, and Propionate, each with GFP, BFP, and TaqRFP). The concentrations of SCFAs used were 0.01 FE, 0.1 FE, 1 FE, 10 FE, 100 FE, and 1000 FE, with 'FE' representing 'Fecal Equivalent.' To see if our constructs can detect SCFA in concentrations that are biologically relevant we measured the SCFA concentration in Fecal Equivalents. Where we calculated the mass of the specific SCFA in 0.01g, 0.1g, 1g, 10g, 100g and 1000g of mouse feces. So, the total mass of a specific short chain fatty acid, that is present in 1g of mouse feces corresponds to 1 Fecal Equivalent. Which then can be scaled up or down. These calculations were done based on the measurements in Shaidullov, Sorokina and Sitdikov3 et al..

As a control, we used the autofluorescence of the bacteria without stimulation to confirm the effect of SCFAs.

It should be noted here that the concentrations greater than 10-100 FE exceed the concentrations of SCFAs that are realistic to achieve in an experimental setup. However, it was interesting to observe the impact of SCFAs on bacteria when exceeding physiological concentrations.

In Figure 8-10, the graphs depicting the fluorescence over a seven-hour time frame are displayed. At first glance, it is noticeable that the fluorescence in the Acetate (Fig. 8) and Butyrate (Fig. 9) constructs increases significantly with higher concentrations compared to lower concentrations. The same trend is observed for Propionate (Fig. 10), but only in the GFP construct. In the case of all GFP constructs, it is worth noting that, when considering the scale, the fluorescence is significantly higher than in the other constructs, while the range remains consistently comparable for both Butyrate and Propionate. However, since the Gibson Assembly did not work, one possible reason for this could be found in the dilutions. We weighed the powdered SCFAs and subsequently created dilution series. If errors were made in the initial weighing, these errors would have propagated through all experiments since we used the same dilutions for all three constructs.

![Second colony PCR](https://static.igem.wiki/teams/4930/wiki/results/acetate.png)

Fig.8: MFI of GLOW.coli over 7 hours after stimulation with acetate.

![Second colony PCR](https://static.igem.wiki/teams/4930/wiki/results/propionate.png)

Fig.9: MFI of GLOW.coli over 7 hours after stimulation with propionate.

![Second colony PCR](https://static.igem.wiki/teams/4930/wiki/results/butyrate.png)

Fig.10: MFI of GLOW.coli over 7 hours after stimulation with butyrate.

Often, it is observed that the fluorescence peak decreases over time, especially at higher concentrations. Occasionally, fluctuations are noticeable, alternating between a peak and nadir. In the future, we intend to conduct the same experiment with the constructs where the Gibson Assembly was successful.

Subsequently, we conducted a literature review and found that certain substances can alter the morphology of bacteria, for example, by damaging the cells and thereby increasing autofluorescence.

## Discussion

A problem we encountered while performing our experiments was the presence of autofluorescence in our sample which distorted our results. Therefore, it is crucial to find ways to minimize or prevent the presence of autofluorescence to ensure the success of future experiments. Solutions for these problems can be found in literature1.

According to *Surre, J., Saint-Ruf, C., Collin, V. et al.*1, prokaryotic cells such as E. coli can exhibit an intrinsic fluorescence due to the presence of fluorescent cellular structural components and metabolites. Examples for these compounds are flavines and NAD, which are responsible for the majority of cytoplasmic autofluorescence in E. coli, since they play an important role in cell metabolism. Both, the fluorescence of FAD and NAD are redox dependent; while the oxidized form of FAD exhibits a strong fluorescence, the reduced form is less fluorescent. For NAD it is the other way around.

Because many fluorescent compounds are present in the cell, cellular autofluorescence encompasses a large spectral range since different endogenous fluorophores emit fluorescence at different wavelengths. Therefore, the autofluorescence spectra oftentimes overlap with the spectra of fluorophores that are introduced for research purposes, hindering fluorescence microscopy and flow cytometry measurements. Generally, the autofluorescence depends on the metabolic state of the cells, as well as on different stressors (e.g.: the use of antibiotics), that affect the cells 1
In their studies, *Surre, J., Saint-Ruf, C., Collin, V. et al.* found that autofluorescence in E.coli increases with bactericidal treatment. For this, β-lactam antibiotic ampicillin was used, leading to an increase in green autofluorescence. Interestingly, this phenomenon could not be observed when a bacteriostatic, protein-synthesis inhibiting antibiotics such as tetracycline were used.Since Carbenicillin, the antibiotic that we used for our experiments, also is a β-lactam antibiotic, another protein-synthesis inhibiting antibiotic needs to be chosen for future experiments to prevent an increase of autofluorescence. For this, the use of another vector that has a resistance to a protein-synthesis inhibiting antibiotic such as tetracycline, might be necessary.

Since the increase of autofluorescence can mostly be attributed to the presence of flavin molecules, and flavine biosynthesis is increased when cells are in severe stress1 (e.g: when a β-lactam antibiotic is used), we expected that the change in antibiotic will lead to a significant decrease of autofluorescence.
Since the autofluorescence observed by *Surre, J., Saint-Ruf, C., Collin, V. et al.* is very similar to the fluorescence spectra of pure flavins, this poses another problem for our experimental set up, specifically, because the emission wavelength of flavines (525 nm)2 is near to the GFP emission wavelength of 510 nm2, and both molecules are excited at similar wavelengths, leading to a spectral overlap.

Lastly, autofluorescence can be further decreased by using PBS instead of cell culture media for the measurements. This is due to the fact that most cell growth media also exhibit fluorescence, since they contain cellular extracts. However, this can be circumvented by the measuring the cells in PBS Buffer instead of LB media or by using M9 medium, for future experiments.

## Outlook

For future research, it would be interesting to investigate the metabolic activity and vitality of bacterial cells after incubation with different concentrations of SCFAs. When considering the gut, there are specific locations with a higher fluid flow than others. For instance, the flow is not as strong in a villus due to its folding. Additionally, there are areas, also known as niches, where certain bacteria are more abundant. Since not all bacteria produce SCFAs to the same extent, the concentration of SCFAs could be concentrated in specific areas of the intestine where the flow is less vigorous, leading to a local increase in concentration. This raises the question of whether the potentially resulting higher concentrations may be toxic to other bacterial species, which in turn could alter the balance in the bacterial flora, favoring an imbalance in SCFAs.

### References

---

[1]Surre, J., Saint-Ruf, C., Collin, V. et al. Strong increase in the autofluorescence of cells signals struggle for survival. Sci Rep 8, 12088 (2018). https://doi.org/10.1038/s41598-018-30623-2

[2] Thermo Fisher GFP data sheet

[3] Shaidullov IF, Sorokina DM, Sitdikov FG, Hermann A, Abdulkhakov SR, Sitdikova GF. Short chain fatty acids and colon motility in a mouse model of irritable bowel syndrome. BMC Gastroenterol. 2021 Jan 26;21(1):37. doi: 10.1186/s12876-021-01613-y. PMID: 33499840; PMCID: PMC7836204.

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