To this date, we provide three different contributions to the 2025 season: (1) Further, advanced tests that can be run on IL10 mutations to ensure its application in therapeutic systems, (2) Statistical analysis plans that can be run on simultaneous factors of temperature and pH conditions, and (3) A database of potential IL10 mutations that have been analyzed by our team and Artificial Intelligence software, including the identification of mutations and potential protein interactions that have been identified on alphafold.

In this cycle of the season, we resolved to work with E. coli (DHα and BL21) in our experiments, with constructed vectors that work accordingly with an E. coli system due to unsuccessful attempts at different bacterial strains. However, we have provided below a viable plan to construct a plasmid and test for its validity in the Lactobacillus plantarum system, often used in probiotics. We hope that this protocol will allow future teams to work with greater ease on a developed project similar in nature; the plans are detailed below.

Our process would involve the construction of a separate plasmid, with the vector backbone sourced from Addgene, pLp_3050sNuc, Plasmid #122030. The sequence map is detailed below, albeit certain aspects beyond the origin of replication and restriction enzyme sites being excluded from the picture.

Upon closer examination, we can see the map image clearer, containing components like sppK and NucA, which would be cut out and replaced by our IL10 sequence using NdeI and EcoRI restriction enzymes. This cloning strategy allows us to integrate our therapeutic payload into a well-characterized backbone.

Our rationale for utilizing sppK stems from its role in regulatory systems in Lactobacillus plantarum. The spp operon is involved in sugar-phosphate stress response, and sppK encodes a histidine kinase that is part of the associated two-component system. By leveraging this native system, we aim to achieve controlled expression of IL-10 in response to environmental cues relevant to gut conditions, thereby improving the therapeutic potential of our engineered strain.

Additional information about the plasmid can also be found on the site, some noteworthy ones being listed below. It is important to note the selectable marker as erythromycin, and the promoter of sppA can be induced by sodium acetate, hence making it accessible as a therapeutic benefit.

After running through a gibson assembly that inserts the sequences to replace NucA with our mutated IL10s, we would run a gel to ensure our correct assembly, then transform our new plasmid into Lactobacillus. After confirming that the Lactobacillus has successfully undergone transformation (grown in Erythromycin), we will induce the system and grow cultures that can run further testing: protein purification and ELISA. Through ELISA, we will ensure that our proteins do not have any adverse effects and retain their functionality while analyzing to determine whether our mutations hold greater stability in greater temperature conditions. We would also include appropriate positive and negative controls to validate the specificity of our cloning and expression system. For example, a wild-type IL-10 control would allow us to compare the bioactivity of our mutated IL-10 variants, while an empty-vector control would confirm that any observed anti-inflammatory effects are due to IL-10 expression and not background Lactobacillus activity.

The following steps are also preferred to be conducted if given the time and budget:

After confirming expression via ELISA, we would perform Western blot analysis to assess protein size and detect potential degradation products, ensuring that the expressed cytokine maintains proper folding. To confirm biological activity, we would culture macrophage or dendritic cell lines with the Lactobacillus supernatant and measure downstream STAT3 phosphorylation through flow cytometry or immunoblotting. This would allow us to confirm that IL-10 is not only being secreted but is also functionally active in triggering the JAK-STAT pathway.

Additionally, we would conduct dose-response experiments by varying sodium acetate concentrations to quantify promoter induction strength and determine whether the sppK-sppA system provides tunable, physiologically relevant expression levels. Long-term stability testing would also be valuable, examining whether Lactobacillus carrying our plasmid maintains IL-10 production across multiple passages, a critical factor for eventual probiotic use. We would also investigate biosafety considerations, including potential off-target effects or horizontal gene transfer risks, by performing co-culture assays with other commensal gut bacteria. Incorporating these steps would strengthen the reliability and translational potential of our work, providing a robust foundation for future therapeutic development using engineered probiotic systems.

While we mainly focused on temperature conditions (in which IL10 is prone to degradation in) this round of experiments, we concluded that it would be preferred to also run tests on the pH levels, stimulating a gut environment that mimics inflammation. As such, statistical analysis would be needed to analyze two independent variables that are simultaneously changing. To ensure the most accurate representation of data, we have collaborated with Dr. Olga Korosteleva to write up an experimental design/statistical analysis that could analyze such results below.

PLANNED STATISTICAL ANALYSIS

Upon completion of data collection, separate two-way ANOVA tests will be conducted for the mutated and non-mutated proteins to evaluate the main effects of temperature and pH, as well as their interaction. Each ANOVA will be based on 5 observations per cell, yielding robust estimates of variability. Following significant effects, post-hoc comparisons will be performed using multiple procedures, including Tukey’s honest significant difference (HSD), and Fisher’s Least Significant Difference (LSD) tests, to identify specific level differences while controlling for Type I error.

To compare the two protein types directly, two-sample t-tests and Wilcoxon rank-sum tests will be conducted at each temperature–pH combination to assess differences in mean concentrations.

Additionally, mean profile plots will be generated separately for temperature and pH, allowing visual comparison of trends between mutated and non-mutated proteins across levels. These graphical summaries will aid in detecting interaction patterns and assessing consistency of effects across protein types.

Schematically mean profiles are depicted below. Plotted will be the means of 25 concentrations with the vertical bars representing plus/minus one standard deviation. The two curves represent two types of proteins. The five points on the x-axis are the five levels for temperature (or pH levels – a different graph).

We have created multiple spreadsheets that detail the mutations that could be altered in IL10 in order to provide the most optimal conditions. Through identification of mutation(s) variant ID, positions, understanding of how the mutations are caused, and explanation of clinical significance, we have compiled a database (though still in progress) of such details.

For the IL10 mutations, our sheet is formatted as follows below. Within each mutation we have also linked relevant articles that credit our research, as well as additional comments which either summarize the information or provide reasoning as to why/why not this mutation is viable in conducting through a wet lab.

One variant we found to be particularly noteworthy was rs1274280163 (position 42, Arg → Stop/Ter) because it introduces a premature stop codon at residue 42, truncating IL-10 early and eliminating ~80% of the protein sequence and critical regions required for receptor (IL-10RA) binding and signaling. The result is a loss-of-function allele: the truncated cytokine cannot form the proper structure or interact with its receptors, which explains the association with severe immune phenotypes such as early-onset inflammatory bowel disease and autoinflammation. Clinically, stop-gained mutations are often highly deleterious because they not only abolish normal protein function but can also trigger nonsense-mediated decay of the mRNA; for IL-10, that loss of anti-inflammatory signaling has clear, mechanistic links to uncontrolled gut inflammation, making this variant both biologically informative and medically important.

Another one that was significant was rs2102441015 (position 34, Pro → Thr) as it replaces proline, a rigid, helix-breaking residue, with threonine, which is more flexible and capable of forming hydrogen bonds. Proline’s cyclic structure often creates kinks in alpha-helices and disrupts secondary structure, so replacing it with threonine may allow IL-10 to adopt a more stable secondary structure and interact more effectively with its receptors. ClinVar lists this variant as being associated with inflammatory bowel disease and of “uncertain significance,” suggesting it could have clinical relevance. Importantly, this mutation could decrease IL-10’s degradability in the intestinal tract and enhance its therapeutic potential by improving structural stability and prolonging its anti-inflammatory effects, making it a promising target for further investigation.

We also chose to study protein interactions to ensure that our mutations would not interfere with any of the IL10’s original relationship to other molecules. As such, we formatted another tab within the sheet to study interactions on Alphafold, noting down factors including: what IL10 binds to, what conditions the binding happens, the interactions involved, a reaction pathway, etc.

While all are important in preventing interference when mutating IL10, the most noteworthy interaction is IL-10 binding to its receptor complex (IL10RA and IL10RB) because it is the critical first step that triggers the entire anti-inflammatory signaling cascade. This interaction occurs on the surface of nearly all IL-10–responsive hematopoietic cells and leads to the activation of JAK1 and TYK2 kinases, which phosphorylate STAT3. Phosphorylated STAT3 then translocates into the nucleus to drive expression of anti-inflammatory genes, suppress antigen presentation, and reduce pro-inflammatory cytokine production. This makes the IL-10/IL10RA/IL10RB interaction central to immune regulation, controlling excessive inflammation that would otherwise lead to tissue damage, colitis, and autoimmune disorders. Its clinical relevance is high, as mutations in IL10RA or IL10RB can cause severe early-onset IBD, demonstrating that when this interaction fails, the immune system loses a key checkpoint for tolerance. Understanding and potentially enhancing this interaction could therefore be a promising therapeutic strategy for inflammatory diseases.

Sources that we used to access the information are also listed on the sheet, including Uniprot and Protein Data Bank Knowledge Base. Additional information on the sheets can be accessed through this link (IL10 Database), including the full version of all research done and the additional sources utilized.