iGEM 2025 — Team concordia-montreal

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block header %}
{% endblock %}
{% block page\_content %}

## ToastBusters Wet Lab Notebook

Our wet lab notebook, **"The Toast-logs"**, was organized as a chronological and collaborative record for the iGEM Concordia team. Each entry is structured by date, team members present, and a clear log of experiments, methods, and results. This consistent format allowed us to systematically track our progress, document troubleshooting—such as optimizing transformations and confirming constructs via sequencing—and record our research into probiotic chassis and enzyme assays. It serves as a complete and practical account of the project's workflow from initial DNA assembly to final testing.

*Note: If you're viewing this page on a mobile device, the embedded PDF may not display fully. For the best experience, view it on a larger screen.*

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block header %}
{% endblock %}
{% block page\_content %}

### Table of contents

* [Wet Lab DBTL](#1)
* [Dry Lab DBTL](#2)
* [References](#3)

## Wet Lab DBTL Cycles

## Cycle 1: Project Ideation and Initial Pivot

#### Design

Our team initially voted to pursue a project targeting the gut–brain axis, aiming to engineer a bacterial probiotic that could contribute to the treatment or prevention of Alzheimer’s disease. We focused on the kynurenine pathway, exploring strategies to upregulate or downregulate metabolites involved in neuroinflammation and neurodegeneration.

#### Build

We began gathering literature and mapping potential genetic targets for metabolic regulation within the kynurenine pathway. We also started developing conceptual models to predict the effects of modulating pathway intermediates on neurological outcomes.

#### Test

As our research deepened, we encountered limited concrete data on feasible genetic targets and measurable outputs. Simulations and literature review revealed that the pathway’s complexity and its dependence on multi-organ interactions would make experimental validation impractical within our scope.

#### Learn

We realized that a project involving the gut–brain axis and neurological endpoints would require extensive multi-system modeling and neuro-assays beyond our available time and resources. Guided by our mentors and Dr. Brett, we decided to pivot while maintaining our focus within the gut microbiome field. This learning led us to redirect our project toward a gut-specific disorder: **Celiac disease**.

---

## Cycle 2: Concept Development

#### Design

After establishing a new focus, we revisited the design phase. Two main branches of thought emerged:

1. **Gluten-Degrading Enzyme Production:** Express a plant- or fungal-derived enzyme within a probiotic bacterium, allowing it to degrade gluten in the lumen of the small intestine into fragments too
   small to trigger immune recognition.
   Our research identified **AN-PEP**, an enzyme previously studied by the 2024 iGEM UT Austin team [1]. Their work provided a valuable starting point, though they
   faced difficulties achieving functional expression in their probiotic strain. We aimed to build upon their findings and overcome those limitations.
2. **Epitope Masking:** A team member proposed an alternative strategy—rather than fully degrading gluten, we could mask the regions recognized by the immune system using analog or binding proteins.

#### Build

We began *in silico* construction of gene parts for gluten degradation and modeled AN-PEP activity on representative gluten sequences. Simultaneously, we conducted literature-based modeling to explore the
feasibility of epitope masking approaches, refining our ideas based on structural information about gluten peptides.

#### Test

Computational modeling revealed that **cleavage alone was not always sufficient**. Depending on sequence variability, some cleavage products remained large enough to retain immunogenic epitopes. Similarly,
early modeling showed that **epitope masking** using analog proteins was biologically complex and difficult to achieve without unintended immune consequences.

#### Learn

We concluded that relying on a single gluten-degrading enzyme might be inadequate for complete detoxification. This led to two refined design directions:

* A **multi-enzyme approach** combining two or more enzymes with complementary cleavage motifs to produce smaller, less immunogenic fragments.
* A modified **epitope-targeting strategy** focusing on blocking or degrading the highly immunogenic **33-mer peptide** region of gluten, as previously explored by the 2024 iGEM UM–Macau team [2].

---

## Cycle 3: *In Silico* Evaluation of AN-PEP Efficiency

#### Design

During the cloning design process, we identified a key question: could AN-PEP degrade gluten efficiently enough to prevent immune detection? If AN-PEP only partially cleaved gluten, the remaining fragments might still trigger immune responses, reducing therapeutic effectiveness.

#### Build

To address this concern, we conducted a computational analysis using our [Software](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27software%27%29%20%7D%7D). The cleavage motif of AN-PEP was input into software
that scanned sequences from multiple gluten strains (compiled from a meta-analysis [link] ). The predicted fragments were then compared against a database of known gluten IgE epitopes in humans. This allowed us
to identify potential overlaps and assess whether AN-PEP could fully eliminate immunogenic regions.

#### Test

Results were encouraging: under idealized conditions, AN-PEP appeared capable of cleaving several gluten variants into non-immunogenic fragments. However, the software did not incorporate kinetic parameters—such
as enzyme concentration, substrate availability, or digestion time—limiting our ability to predict real-world performance. These findings indicated the need for future **wet-lab experiments** to test enzymatic
activity and degradation rates.

#### Learn

We concluded that while AN-PEP is a strong candidate enzyme, relying solely on it introduces risk. Implementing a **multi-enzyme system** could improve degradation efficiency by acting on diverse cleavage motifs,
generating smaller fragments, and reducing the probability of leaving intact epitopes. This insight shaped the direction of our next design cycle, focusing on enzyme combination strategies.

---

## Cycle 4: Cloning and Wet-Lab Validation of AN-PEP

#### Design

Following the computational validation of AN-PEP, we proceeded to clone the construct using the Golden Gate assembly method.
The plasmid backbone used in this procedure contains a fluorescent green cassette that is excised when successfully digested by BsaI type II restriction
enzymes. Therefore, successful clones were expected to appear white, indicating the likely presence of the assembled AN-PEP plasmid.

However, as detailed in the Sequencing section of our Wet Lab Experiments page, sequencing results from several white colonies revealed unexpected gaps of 1–1.5 kb precisely where the AN-PEP gene, promoter, ribosome binding site (RBS), secretion tag, and terminator should have been. This discrepancy suggested either incomplete digestion or errors during synthesis or assembly. To evaluate a larger number of colonies efficiently and cost-effectively, we designed a secondary screening approach to assess insert presence by plasmid size.

#### Build

We performed plasmid mini-preps on selected colonies and used 1% agarose gel electrophoresis to compare plasmid sizes.
To minimize the interference of supercoiled plasmid conformations, which can obscure accurate size estimation, each
sample was digested with XbaI prior to loading on the gel. This allowed for clearer visualization and more reliable comparison between expected and observed plasmid sizes.

#### Test

We analyzed eight colonies from each of the four plates (though we ran a total of 20 colonies). After electrophoresis, none of the samples displayed plasmid sizes consistent with the predicted
construct length. This consistent discrepancy across all plates strongly indicated that the AN-PEP insert was not successfully incorporated.

#### Learn

The absence of the correct-sized plasmid across multiple clones suggested a systematic failure in the cloning process rather than random colony variation. Potential causes include incomplete
restriction digestion, ligation inefficiency, or synthesis errors within the AN-PEP fragment itself. This outcome highlighted the need to re-evaluate our assembly protocol and consider alternate
cloning strategies, such as Gibson Assembly or re-synthesizing the AN-PEP insert, to ensure successful plasmid construction. To avoid time loss, the full transcription unit was synthesized.

---

## Cycle 5:

#### Design

The full transcriptional unit (TU) was designed to match the configuration planned for the final construct, with BsaI cut sites removed to prevent interference during future Golden Gate assemblies. The TU
included, in order: a promoter, ribosome binding site (RBS), secretion tag, the AN-PEP enzyme, and a terminator sequence. This complete expression cassette was synthesized and inserted into a blank pET
backbone containing kanamycin resistance as the selection marker.

#### Build

Once the construct was received, we began transformation experiments to introduce the AN-PEP TU plasmid into *E. coli* strains. Transformations were attempted in both BL21 and *E. coli* Nissle
1917, as we were concurrently preparing these strains to express in the secondary part of our project working with the peptide caps.

#### Test

Following transformation, no growth was observed on the BL21 selection plates. However, colonies successfully appeared on the Nissle 1917 plates, indicating successful transformation in that host strain.
Given this positive result, subsequent work focused on the Nissle strain for downstream characterization.

#### Learn

The successful transformation of *E. coli* Nissle 1917 suggests it is a suitable chassis for AN-PEP. The next steps will involve confirming expression of the AN-PEP enzyme and peptide caps in Nissle
and proceeding to functional assays to assess enzymatic activity and gluten degradation efficiency.

---

## Dry Lab DBTL Cycles

## Cycle 1: Streamlining Software Use

#### Design

There were two possible designs for the software. One combined all functionalities into a single program, the other had one script for each function. We wanted to ensure our program was convenient for the
end user. Using one program is more convenient. However, ensuring reasonable run times is also an important quality of life consideration. We needed to determine which setup was best for the end user.

#### Build

To determine which design was most convenient both designs were created in R using the R-studio console and tested on our database - GlutPro 6.1 [3].

#### Test

The programs were compared for speed and efficiency of use. Due to the large amount of data intended to be processed the program with the different functions divided into different files performed
significantly faster than the combined program.

#### Learn

The reduced time requirement of the program built with separate scripts was determined to be a better quality of life feature than having all the programs in one file. As such it was this version of the
program that was carried through into future versions.

---

## Cycle 2: The Addition of a Positive Control

#### Design

Through many smaller DBTL cycles the code had been brought to a point where the enzymes could cut the sequences, epitopes could be matched and output was presented in a series of files - in effect the code
fully met the requirements of the minimum viable product. What remained unsure was the accuracy of the code.

#### Build

To determine if the code was performing accurately a positive control was created. Running this code along with the results would provide confidence to the users that their results were accurate.

#### Test

The positive control functioned as intended and has since its inception returned positive results.

#### Learn

The inclusion of the positive control demonstrated that our code was accurate and could be used as a model for epitope binding to enzyme fragments.

## References

1. UT Austin iGEM 2024. (2024). Igem.wiki. https://2024.igem.wiki/austin-utexas/results
2. UT-Macau. (2024). Igem.wiki. https://2024.igem.wiki/um-macau/results
3. Bromilow, S., Daly, M., Gethings, L. A., Mills, E. N. C., Nitride, C., & Shewry, P. R. (2020). The GluPro suite of curated cereal seed storage prolamin protein sequence datasets [Data set]. figshare. https://doi.org/12613154

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block header %}
{% endblock %}
{% block page\_content %}

### Table of contents

* [Bacteria](#bacteria)
* [Enzymes](#enzymes)
* [Peptides](#peptides)
* [Assays](#assays)
* [Sequencing](#sequencing)
* [References](#ref)

## Bacteria

To achieve our goal of developing a long-term, **low-stress solution to gluten cross-contamination**, selecting a suitable probiotic host was critically important.
The ideal bacterium had to be safe for human consumption (Generally Recognized As Safe, GRAS), capable of colonizing the gut, resilient under gastrointestinal conditions, and able to efficiently produce and secrete enzymes.

For context, one enzyme-based supplement available in Canada is *GluteGuard*, which contains caricain, a papaya-derived enzyme that can help reduce symptoms of accidental gluten exposure [1].
However, this supplement must be taken before every meal, placing a significant burden on users. Our aim with a probiotic host was to provide continuous protection in the gut, allowing the bacteria to act throughout the day while requiring only a single daily dose.

### Initial Cloning and Proof of Concept

For initial development, cloning was performed in ***Escherichia coli DH5α*** due to its genetic stability and reliability for molecular cloning [2].
As a model organism, *E. coli* was ideal for proof-of-concept studies due to its well-characterized genetics and competence.

However, choosing a gram-negative bacterium like *E. coli* introduced technical challenges. Unlike gram-positive bacteria, which are more commonly used in probiotics and are naturally better at secreting proteins, *E. coli* has an outer membrane that limits secretion efficiency.
These challenges required careful consideration of secretion tags, promoter sequences, and selection markers, with ampicillin or kanamycin resistance used to maintain stable cultures.

### Considering Alternative Chassis

The team explored several alternative probiotic chassis, including gram-positive strains such as *Lactiplantibacillus plantarum*, which are generally recognized for their higher secretion efficiency.
Switching to a gram-positive chassis would have required redesigning the genetic constructs, including promoters, secretion tags, ribosome-binding sites, and selection markers.
Given the project timeline, this approach was considered too time-intensive and risky.

In parallel, the team explored isolating strains from the probiotic blend Webber Natural Digestion Probiotic 30 Billion containing eight different strains. Initial inoculation and plating produced a dense bacterial lawn, which later serial dilutions allowed for some distinguishable colonies.
Differentiating all eight strains without strain-specific selective media proved too complex and time-consuming, so this part of the project was eventually deprioritized.
The effort did, however, inform future considerations for selective agents and experimental design.

The tables below summarize the **strains considered**, outlining the advantages and disadvantages of each potential probiotic chassis.

**Table 1.** Advantages and disadvantages of using *Escherichia Coli* Nissle 1917 as a probiotic chassis [3-9].

| *Escherichia Coli* | |
| --- | --- |
| Advantages | Disadvantages |
| Wide use as a probiotic CRISPR tools | Genotoxic compound: colibactin (links to colorectal cancer) |
| Genetic tractability (decades of studies) | Induce DNA damage in colon cells |
| Gram Negative, better for our DH5 alpha (expression) | Potential horizontal gene transfer risk |
| Gut colonization potential |  |

**Table 2.** Advantages and disadvantages of using *Lactiplantibacillus plantarum* as a probiotic chassis [10-17].

| *Lactiplantibacillus plantarum* | |
| --- | --- |
|| Advantages | Disadvantages |
| --- | --- |

| Strain TWK10 shows no questionable effects | Strain-specific safety issues (risk in immunocompromised patients) |
| Strong gut survivability and GRAS status | Gram-positive (different cell wall properties than *E. coli*) |
| Wide use as a probiotic with well-established results | Lower transformation efficiency compared to *E. coli* Nissle 1917 |
| Non-pathogenic background | Fewer available genetic tools compared to *E. coli* Nissle 1917 |

**Table 3.** Advantages and disadvantages of using *Bifidobacterium longum* subsp. *longum* BL21 as a probiotic chassis. [18-23]

| *Bifidobacterium longum* | |
| --- | --- |
|| Advantages | Disadvantages |
| --- | --- |

| GRAS status | Gram-positive (different secretion properties than *E. coli*) |
| Shown to reduce gliadin-induced toxicity | Challenging genetic manipulation and low transformation efficiency |
| Anti-inflammatory effects and anaerobic metabolism | Fewer available genetic tools than *Lactobacillus* or *E. coli* Nissle 1917 |
| Commonly used as a probiotic species | Strictly anaerobic, requiring specialized growth conditions |

**Table 4.** Advantages and disadvantages of using *Lacticaseibacillus rhamnosus* GG as a probiotic chassis [24-28].

| *Lacticaseibacillus rhamnosus* | |
| --- | --- |
| Advantages | Disadvantages |
| Extensively studied and proven safe for gut function | Gram-positive, with lower protein expression levels compared to *E. coli* Nissle 1917 |
| Strong acid and bile tolerance with high adhesion capacity | Possible risk of sepsis or bacteremia in immunocompromised patients |
| Ability to form stable biofilms in the intestinal environment | Biofilm formation enhanced in glucose-rich conditions, which may complicate containment or control |

### Final Choice

Ultimately, the team decided to remain with the *E. coli*, selecting ***E. coli* Nissle 1917**, a strain with a long history of safe human use.
This choice required addressing specific technical considerations: designing type-specific secretion tags for extracellular proteins, choosing compatible promoter sequences and selection markers, and preparing optimized media for *E. coli* physiology.
Although other gram-positive strains were evaluated, *E. coli* Nissle 1917 provided the best balance between safety, feasibility, and project constraints.

## Enzymes

Working towards our goal of engineering this probiotic, we initially searched for enzymes capable of targeting gluten or its analogs. Our review of enzyme treatments for Celiac disease revealed several promising candidates for oral enzyme therapy.
For our project, we focused on AN-PEP, a prolyl endopeptidase derived from *Aspergillus niger*, while the evaluation of other enzymes was performed entirely in silico.
At first, we examined these enzymes individually, but as we progressed in our research, we began exploring combinations of complementary enzymes to potentially enhance gluten degradation efficiency (see below).

#### AN-PEP

Prolyl endopeptidase (PEP) derived from *Aspergillus niger* (AN-PEP) has been shown to effectively degrade immunogenic gluten peptides. In a dynamic, multi‐compartmental gastrointestinal in vitro model, AN‐PEP was shown to degrade almost all immunogenic gluten epitopes from gluten‐containing meals into non‐immunogenic
fragments during passage through the stomach compartment [29].

#### FVp-P

Fvp-P, a gluten-degrading enzyme from *Flammulina velutipes*, demonstrated the ability to cleave all Celiac disease–active peptides at multiple sites with greater efficiency than AN-PEP.
These findings indicate that Fvp-P represents a promising candidate for gluten degradation in real food matrices [30].
The enzyme was also utilized by the Macau iGEM team in 2023 [31], where it showed the most promising performance among the enzymes tested.

#### Kuma030

Kuma030 is a rationally designed synthetic enzyme developed to specifically degrade immunogenic gluten peptides.
One study showed that treatment of gliadin with Kuma030 eliminates its ability to trigger a T cell response. In simulated gastric digestion assays, Kuma030 degraded over 99% of the immunogenic gliadin fraction within physiologically relevant time frames, reducing it below the toxic threshold for celiac patients and demonstrating strong potential as an oral therapeutic candidate for Celiac disease [32].
It was also evaluated by the Macau iGEM team in 2023 [31], although they were unable to successfully express the enzyme.

#### EP-B2 and SC-PEP

EP-B2, a glutamine-specific cysteine endoprotease from germinating barley seeds, and SC-PEP, a prolyl endopeptidase from *Sphingomonas capsulata*, have been studied together as a combination therapy for Celiac disease.
SC-PEP specifically cleaves the proline residues. In vitro and in vivo evaluation of this enzyme combination in rats demonstrates its ability to retain its enzymatic activity in acidic environments comparable to the stomach and duodenum [33].
However, a phase II clinical trial later showed that the combination did not significantly improve histologic or symptom scores compared to placebo, likely due to limited enzymatic activity and degradation within the gastric environment [34].

#### EP-B2 and AN-PEP

The combination of EP-B2 and AN-PEP offers the advantage of maintaining enzymatic activity and stability in the acidic environment of the stomach.
In this pair, EP-B2 initially cleaves large gluten proteins at glutamine residues, producing smaller oligopeptides that can still trigger inflammation.
AN-PEP then further hydrolyzes these peptides at proline residues, effectively breaking down gliadin into non-immunogenic fragments [35].

## Peptides

Gliadin, one of the two major protein components of gluten, is a key trigger of the immune response in individuals with celiac disease. This protein contains several epitope-binding regions that can provoke adverse immune reactions. To address this, we considered developing a peptide capable of shielding or blocking these reactive sites as a potential strategy to mitigate gluten’s immunogenic effects [36].

Our next step was to identify peptides that could bind to the immunogenic 33-mer amino acid region of the gliadin sequence—potentially preventing recognition of its epitope-rich areas. Previous research using phage display libraries (12-mer and 7-mer amino acids) identified over 160 unique gliadin-binding peptide sequences.
Among these, **p61 and p64** were the most frequently isolated peptides [37]. These peptides could function either independently or in conjunction with gluten-degrading enzymes.

**Table 5.** Frequency of peptide observations(p61 and p64). The observation frequency directly reflects their binding specificity and affinity toward the gliadin protein [37].

| Peptide | Sequence | Frequency (%) |
| --- | --- | --- |
| P61 | WHWRNPDFWYLK | 22.5 |
| P64 | WHWTWLSEYPPP | 21.5 |

## Assays

To test our project, we selected three complementary assays to evaluate the **enzymatic breakdown of gluten.** Additionally, we implemented two assays to verify that our **peptide caps functioned as intended**.

### Enzymatic breakdown of gluten

#### Gluten and Gliadin Plate Assays The first two assays utilized agar plates containing either gluten or gliadin. Both operated on the same principle—assessing proteolytic activity, specifically the ability of our expressed enzymes to degrade gluten or gliadin, as indicated by the formation of a clear zone around bacterial colonies. To perform the assay, transformed *E. coli* cultures were plated with IPTG to induce the lac operator, triggering expression and secretion of the target enzymes. This induction allowed colonies capable of degrading the substrate to form visible **zones of clearance.** Although gluten plates are typically used to identify bacteria that naturally degrade gluten, in our case they served to confirm that our plasmid-encoded gluten-degrading enzyme was functioning as expected. *E. coli* strains cannot degrade gluten or gliadin naturally without a genetically modified plasmid. Previous studies have identified several bacterial species with inherent gluten-degrading capabilities, including *Streptococcus salivarius*, *Bacillus pumilus*, *Rothia dentocariosa*, *Bacillus subtilis*, and *Staphylococcus epidermidis*—all isolated from human saliva [38]. These could serve as positive controls, with *Bacillus subtilis* being the most accessible candidate for use in Level 1 microbiology labs. Negative controls consisted of untransformed *E. coli* strains such as *DH5α*, which lack gluten-degrading capacity. Colorimetric AN-PEP Activity Assay The third assay was a colorimetric assay adapted from the method of [39]. It measures the activity of the enzyme AN-PEP by monitoring optical density at 410 nm during incubation with gluten and Z-Gly-Pro-pNA (5 mM) in K phosphate buffer (pH 7.0, 0.1 M). The reaction is stopped using acetate buffer (pH 4.0, 1 M) containing 10% Triton X-100, with a final volume of 250 µL per well in a 96-well plate. Z-Gly-Pro-pNA is a synthetic substrate of prolyl endopeptidases; upon cleavage, it releases a yellow chromophore measurable at 410 nm. This assay allowed us to evaluate two variables: * **Degradation time:** Determining the time required for AN-PEP to reduce gluten concentration to a “safe” threshold. Reactions were run at 5-minute intervals for 95 minutes, informed by our survey results indicating most individuals with celiac disease experience symptoms within 0–3 hours of gluten exposure. * **Degradation efficiency:** Measuring gluten degradation at varying initial concentrations (1000 ppm to 10 ppm). The lower value was selected after consultation with Dr. Therrien, considering that the Canadian standard for gluten-free products is below 20 ppm [40]. Our aim was to establish standard curves using the commercial probiotic GluteGuard before testing our own enzyme AN-PEP, allowing for a direct comparison of degradation efficiency between our probiotic and products currently available on the market. Peptide Cap Evaluation ELISA The second set of assays aimed to evaluate the effectiveness of the peptide caps using ELISA-based technology. An **ELISA (enzyme-linked immunosorbent assay)** is a biochemical technique that detects the presence of a target molecule—such as a protein or antigen—through specific antibody binding, producing a measurable color change. In this context, ELISA detects gluten or gliadin based on their ability to bind specific antibodies. If our peptide caps successfully masked the epitope regions of gliadin, the antibodies would bind less effectively, resulting in a negative or reduced signal. Due to time and financial constraints, a full quantitative ELISA kit could not be obtained. Instead, a commercially available rapid gluten detection test—capable of identifying gluten concentrations as low as 10 ppm—was used [41]. Although qualitative, this test provided a fast and affordable preliminary assessment, allowing us to gauge the potential effectiveness of the peptide caps while simulating the experience of someone living with celiac disease. His-Tag Synthesis and Purification Both the enzymes and peptide caps were synthesized to include a His-tag, enabling potential enzyme activity assays with purified samples. The His-tag, a short sequence of histidine residues, allows rapid and efficient purification of the target protein from a mixture of cellular proteins through **immobilized metal affinity chromatography (IMAC)**. Nickel-binding beads selectively interact with the histidine residues, washing away unwanted proteins and permitting the elution of a highly purified enzyme under controlled conditions. These purified samples are suitable for downstream analyses, including activity assays or structural studies.

## Sequencing

Samples for sequencing included both parts from the iGEM distribution kit and synthesized parts ordered from Twist Bioscience. The team used the Monarch Spin Plasmid Miniprep Kit (NEB T1110) protocol [42].
During the first few sequencing attempts, we encountered issues with low plasmid DNA concentrations, particularly with the distribution kit parts.
After learning that several other iGEM teams were experiencing similar difficulties, we implemented a series of optimization steps to improve yield:

* **Increased culture volume:** Using the largest recommended overnight culture volume of 5 mL for mini preps.
* **Sample concentration:** After centrifugation, we removed most of the supernatant and resuspended the remaining pellet in approximately 100 µL of solution to maximize cell density.
* **Reduced elution volume:** DNA was eluted in only 30 µL to achieve higher concentration.

Despite these adjustments, some samples remained at the lower end of the acceptable concentration range. However, we opted to send slightly larger total volumes for sequencing, as this would facilitate the process even if concentrations were below the optimal range. We were pleased that the sequencing results for most of our samples came back as expected.

All parts were initially designed and verified *in silico* using SnapGene through simulated Golden Gate cloning.
Once sequencing results were received, alignment analysis in SnapGene allowed us to easily confirm that our cloned sequences matched the expected constructs.
In a few cases, minor variations such as single-nucleotide substitutions were observed, but since these occurred outside of our parts of interest, they were not considered problematic.

Both transformed parts and Golden Gate–assembled clones were sent for sequencing through **Plasmidsaurus.**
Overall, sequencing verification confirmed that our assembly and cloning strategies were effective, validating both the design and the experimental workflow used throughout this stage of the project.

### Troubleshooting AN-PEP Cloning and Unexpected Deletions

The cloning process proved crucial in uncovering a significant issue with the AN-PEP gene.
During cloning using the **Golden Gate assembly protocol**, we employed **green–white screening**, as our plasmid backbone contained a fluorescent green cassette.
Following successful cloning, this cassette would be replaced with our part insert.

When we first checked the transformation plates, we were thrilled to observe small white colonies—an early indication that cloning may have been successful.
However, to confirm that the AN-PEP gene was properly inserted, two white colonies from each plate were picked, grown overnight in liquid culture, and mini-prepped.
The DNA concentrations from these preps were satisfactory, and the samples were subsequently sent for sequencing.

The sequencing results, however, were not what we expected.
In all eight of the samples, the **AN-PEP insert was missing**, with an approximately **1.5 kb deletion** occurring in the region where the gene should have been.
In most cases, the promoter, ribosome binding site (RBS), and terminator were also partially or completely deleted along with the AN-PEP sequence.
The deletions varied slightly in both position and length, creating considerable confusion.

Adding to the uncertainty, one of the eight samples showed a plasmid size of approximately **7 kb**, compared to the expected **4 kb**, while the remaining samples were closer to **3 kb.** We hypothesized that the larger plasmid might have resulted from an unexpected plasmid fusion event, though the mechanism behind this and its connection to the deletions remained unclear.

Our mentors suggested that the correct clone might still be present among our colonies and encouraged us to continue sampling to locate an intact version. Nonetheless, several members of the team argued that since all **eight samples exhibited nearly identical deletions**, and similar issues had reportedly been observed by the UT Austin iGEM team, the problem was likely linked to the coding sequence of AN-PEP itself or its behavior during cloning.

To further investigate, we performed agarose gel electrophoresis to visualize the plasmid sizes. Knowing the expected plasmid length, we digested the samples prior to running the gels to minimize the presence of supercoiled plasmids, which can complicate accurate size determination.

This phase of troubleshooting provided valuable insights into how certain coding sequences—particularly large or complex ones like AN-PEP which has multiple repetitive codon sequeces—may behave unpredictably during cloning. It also highlighted the importance of verifying results through multiple methods, including sequencing and gel electrophoresis, before drawing conclusions about cloning success.

After weeks of trying to isolate a clone with our designed plasmid without success, we opted to instead order the full transcription unit believing that it would be more time efficient to move forward with the project rather than continue to somewhat blindly test every possible colony.

## References

1. Canadian Celiac Association, & Vlasic, L. (2024, November 21). GluteGuard - Celiac Canada. Celiac Canada. https://www.celiac.ca/gluteguard/
2. DH5α Competent Cells - US. (n.d.). Www.thermofisher.com. https://www.thermofisher.com/ca/en/home/life-science/cloning/competent-cells-for-transformation/competent-cells-strains/dh5a-competent-cells.html
3. Falzone, L., Lavoro, A., Candido, S., Salmeri, M., Zanghì, A., & Libra, M. (2024). Benefits and concerns of probiotics: an overview of the potential genotoxicity of the colibactin-producing *Escherichia coli* Nissle 1917 strain. *Gut Microbes, 16*(1). https://doi.org/10.1080/19490976.2024.2397874
4. Hu, R., Lin, H., Li, J., Zhao, Y., Wang, M., Sun, X., Min, Y., Gao, Y., & Yang, M. (2020). Probiotic *Escherichia coli* Nissle 1917-derived outer membrane vesicles enhance immunomodulation and antimicrobial activity in RAW264.7 macrophages. *BMC Microbiology, 20*(1). https://doi.org/10.1186/s12866-020-01953-x
5. Massip, C., Branchu, P., Bossuet-Greif, N., Chagneau, C. V., Gaillard, D., Martin, P., Boury, M., Sécher, T., Dubois, D., Nougayrède, J.-P., & Oswald, E. (2019). Deciphering the interplay between the genotoxic and probiotic activities of *Escherichia coli* Nissle 1917. *PLOS Pathogens, 15*(9), e1008029. https://doi.org/10.1371/journal.ppat.1008029
6. Nougayrède, J.-P., Chagneau, C. V., Motta, J.-P., Bossuet-Greif, N., Belloy, M., Taieb, F., Gratadoux, J.-J., Thomas, M., Langella, P., & Oswald, E. (2021). A Toxic Friend: Genotoxic and Mutagenic Activity of the Probiotic Strain *Escherichia coli* Nissle 1917. *MSphere, 6*(4). https://doi.org/10.1128/msphere.00624-21
7. Scaldaferri, F., Gerardi, V., Mangiola, F., Lopetuso, L. R., Pizzoferrato, M., Petito, V., Papa, A., Stojanovic, J., Poscia, A., Cammarota, G., & Gasbarrini, A. (2016). Role and mechanisms of action of *Escherichia coli* Nissle 1917 in the maintenance of remission in ulcerative colitis patients: An update. *World Journal of Gastroenterology, 22*(24), 5505. https://doi.org/10.3748/wjg.v22.i24.5505
8. Shaughnessy, A. F. (2005). Probiotic Useful for Preventing Relapse of Ulcerative Colitis. *American Family Physician, 71*(10), 1990–1992.
9. Sonnenborn, U. (2016). *Escherichia coli* strain Nissle 1917—from bench to bedside and back: history of a special *Escherichia coli* strain with probiotic properties. *FEMS Microbiology Letters, 363*(19), fnw212. https://doi.org/10.1093/femsle/fnw212
10. Blanch‐Asensio, M., Dey, S., Varun Sai Tadimarri, & Sankaran, S. (2023). Expanding the genetic programmability of *Lactiplantibacillus plantarum*. *Microbial Biotechnology, 17*(1). https://doi.org/10.1111/1751-7915.14335
11. Goossens, D., Jonkers, D., Russel, M., Thijs, A., Bogaard, A. van den, Stobberingh, E., & Stockbrügger, R. (2005). Survival of the probiotic, *L. plantarum* 299v and its effects on the faecal bacterial flora, with and without gastric acid inhibition. *Digestive and Liver Disease, 37*(1), 44–50. https://doi.org/10.1016/j.dld.2004.07.018
12. Карасева, О. В., Ozhegov, G. D., Dilyara Khusnutdinova, Siniagina, M., Anisimova, E. A., Farida Akhatova, Rawil Fakhrullin, & Yarullina, D. R. (2023). Whole Genome Sequencing of the Novel Probiotic Strain *Lactiplantibacillus plantarum* FCa3L. *Microorganisms, 11*(5), 1234–1234. https://doi.org/10.3390/microorganisms11051234
13. Lu, S., Yue, K., He, S., Huang, Y., Ren, Z., & Xu, J. (2025). Safety Assessment of *Lactiplantibacillus plantarum* GUANKE Based on Whole-Genome Sequencing, Phenotypic, and Anti-Inflammatory Capacity Analysis. *Microorganisms, 13*(4), 873–873. https://doi.org/10.3390/microorganisms13040873
14. Mugwanda, K., Hamese, S., Van Zyl, Winschau F., Prinsloo, E., Du Plessis, M., Dicks, Leon M. T., & Thimiri Govinda Raj, Deepak B. (2023). Recent advances in genetic tools for engineering probiotic lactic acid bacteria. *Bioscience Reports, 43*(1). https://doi.org/10.1042/bsr20211299
15. Ozawa, Y., Hara, M., Shimohira, M., Sakurai, K., Nakagawa, M., & Shibamoto, Y. (2016). Associations between computed tomography features of thymomas and their pathological classification. *Acta Radiologica, 57*(11), 1318–1325. https://doi.org/10.1177/0284185115590288
16. Singh, R. P., Shadan, A., & Ma, Y. (2022). Biotechnological Applications of Probiotics: A Multifarious Weapon to Disease and Metabolic Abnormality. *Probiotics and Antimicrobial Proteins, 14*(6). https://doi.org/10.1007/s12602-022-09992-8
17. Umanets, A., Surono, I. S., & Venema, K. (2023). I am better than I look: genome based safety assessment of the probiotic *Lactiplantibacillus plantarum* IS-10506. *BMC Genomics, 24*(1). https://doi.org/10.1186/s12864-023-09495-y
18. Guo, H., Fan, M., Hou, T., Li, Y., Wang, S., Wang, X., Peng, H., Wang, M., Wu, T., & Zhang, Y. (2023). Efficacy and Safety of *Bifidobacterium longum* Supplementation in Infants: A Meta-Analysis of Randomized Controlled Trials. *Foods, 12*(24), 4451–4451. https://doi.org/10.3390/foods12244451
19. Matera, M. (2024). BIFIDOBACTERIA, LACTOBACILLI….When, How and Why should we use them. *Global Pediatrics, 8*, 100139–100139. https://doi.org/10.1016/j.gpeds.2024.100139
20. O’Callaghan, A., & van Sinderen, D. (2016). Bifidobacteria and Their Role as Members of the Human Gut Microbiota. *Frontiers in Microbiology, 7*(925). https://doi.org/10.3389/fmicb.2016.00925
21. Roda, C., Mahmoud, O., Peralta, G. P., Fuertes, E., Granell, R., Serra, I., Henderson, J., Jarvis, D., & Garcia-Aymerich, J. (2019). Physical-activity trajectories during childhood and lung function at 15 years: findings from the ALSPAC cohort. *International Journal of Epidemiology, 49*(1), 131–141. https://doi.org/10.1093/ije/dyz128
22. Scherer, T. C., Marco, I. D., Coldebella, R., Pimentel, T. C., Magnani, M., Guilherme, Bagolin, A., & Silvani Verruck. (2025). Bifidobacteria and Celiac Disease: Mechanisms of Probiotic Action in Reducing Gluten‐Induced Cytotoxicity and Inflammation. *Molecular Nutrition & Food Research*. https://doi.org/10.1002/mnfr.70222
23. Skoufou, M., Tsigalou, C., Stergios Vradelis, & Bezirtzoglou, E. (2024). The Networked Interaction between Probiotics and Intestine in Health and Disease: A Promising Success Story. *Microorganisms, 12*(1), 194–194. https://doi.org/10.3390/microorganisms12010194
24. Eze, U. J., Lal, A., Elkoush, M. I., Halytska, M., & Syed Atif. (2024). Recurrent *Lactobacillus rhamnosus* Bacteremia and Complications in an Immunocompromised Patient With History of Probiotic Use: A Case Report. *Curēus*. https://doi.org/10.7759/cureus.54879
25. Mathipa-Mdakane, M. G., & Thantsha, M. S. (2022). *Lacticaseibacillus rhamnosus*: A Suitable Candidate for the Construction of Novel Bioengineered Probiotic Strains for Targeted Pathogen Control. *Foods, 11*(6), 785. https://doi.org/10.3390/foods11060785
26. Mugwanda, K., Hamese, S., Van Zyl, Winschau F., Prinsloo, E., Du Plessis, M., Dicks, Leon M. T., & Thimiri Govinda Raj, Deepak B. (2023). Recent advances in genetic tools for engineering probiotic lactic acid bacteria. *Bioscience Reports, 43*(1). https://doi.org/10.1042/bsr20211299
27. Schuh, A., & Schmickal, T. (2019). Clavicle fracture. *MMW Fortschritte Der Medizin, 161*(1), 47. https://doi.org/10.1007/s15006-019-0068-9
28. Segers, M. E., & Lebeer, S. (2014). Towards a better understanding of *Lactobacillus rhamnosus* GG - host interactions. *Microbial Cell Factories, 13*(Suppl 1), S7. https://doi.org/10.1186/1475-2859-13-s1-s7
29. Salden, B., et al. "Randomised clinical study: gluten degradation with enzyme combination in patients with coeliac disease."Alimentary Pharmacology & Therapeutics, 42(11-12), (2015).
30. Ersoy, B., et al. “Novel approaches in gluten degradation: implications for celiac disease therapy.”Frontiers in Immunology, 14, (2023).
31. Wolf, C., et al. “Enzymatic degradation of gluten: Implications for celiac disease treatment.”Digestive and Liver Disease, 47(9), (2015).
32. Parts | UM-Macau - iGEM 2023. (2023). Igem.wiki. https://2023.igem.wiki/um-macau/parts
33. Stoven, S., et al. “Gluten-degrading enzymes for celiac disease: in vitro and in vivo evaluation.”Digestive Diseases and Sciences, 57(10), (2012).
34. Xiao, R., et al. “Microbial enzymes for gluten hydrolysis and potential applications in celiac disease therapy.”Applied Microbiology and Biotechnology, 104(5), (2020).
35. Gass, J., et al. “Combining prolyl endopeptidase and cysteine endoprotease: a gluten detoxification strategy for celiac sprue.”Gut, 56(2), (2007).
36. Barone, M. V., Troncone, R., & Auricchio, S. (2014). Gliadin Peptides as Triggers of the Proliferative and Stress/Innate Immune Response of the Celiac Small Intestinal Mucosa. *International Journal of Molecular Sciences, 15*(11), 20518–20537. https://doi.org/10.3390/ijms151120518
37. Chen, T., Hoffmann, K., Östman, S., Sandberg, A.-S., & Olsson, O. (2011). Identification of gliadin-binding peptides by phage display. *BMC Biotechnology, 11*, 16. https://doi.org/10.1186/1472-6750-11-16
38. Berger, M., Sarantopoulos, C., Ongchangco, D., Sry, J., & Cesario, T. (2015). Rapid isolation of gluten-digesting bacteria from human stool and saliva by using gliadin-containing plates. *Experimental Biology and Medicine (Maywood, N.J.), 240*(7), 917–924. https://doi.org/10.1177/1535370214564748
39. Liu, M., et al. (2023). High-level production of *Aspergillus niger* prolyl endopeptidase from agricultural residue and its application in beer brewing. *Microbial Cell Factories, 22*(1). https://doi.org/10.1186/s12934-023-02087-1
40. Canadian Food Inspection Agency. (2024). Compliance and enforcement of gluten-free claims - inspection.canada.ca. Canada.ca. https://inspection.canada.ca/en/food-labels/labelling/industry/allergens-and-gluten/gluten-free-claims
41. EZ Gluten. (n.d.). EZ Gluten – Gluten Testing – Food Testing. *EZ Gluten*. Retrieved October 6, 2025, from https://ezgluten.com/
42. New England Biolabs. (2025). Neb.com. https://www.neb.com/en-ca/products/t1110-monarch-spin-plasmid-miniprep-kit

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block header %}
{% endblock %}
{% block page\_content %}

### Table of contents

* [Results](#results)
* [Suplemental](#suplemental)

## Results

Initial screening for the successful transformation of *E. coli* DH5ɑ with plasmids containing AN-PEP and different secretion tags (Dsba1, MalE, TorA or no-tag) was performed by selecting colonies that exhibited GFP fluorescence (Figure 1).

![GFP colonies plate](https://static.igem.wiki/teams/5785/results/results-gfp.webp)

**Figure 1.** Bacterial cultures of *E. coli* inoculated with plasmids containing AN-PEP and different secretion tags, screened for transformation via GFP.

Four plates were prepared for the three secretion tags (DsbA, MalE, and TorA) and a no-tag control. As shown in Figure 2, white colonies (circled) were selected for testing, with numbered labels used to ensure consistent sample identification.

![Plate results](https://static.igem.wiki/teams/5785/results/plates-results.webp)

**Figure 2.** Golden Gate Cloning of AN-PEP Constructs (same plates as Figure 1).

Plasmid DNA was isolated from GFP-positive colonies, and its purity was assessed by spectrophotometry.
As shown in Table 1 (supplemental), the 260/280 absorbance ratios for most samples were approximately 1.8, indicating minimal protein contamination.
However, the DNA concentrations—particularly during the initial extractions—were noticeably low.

Following protocol optimizations described in the [Sequencing](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#sequencing) section, later plasmid mini preps yielded higher DNA concentrations, providing sufficient plasmid for downstream applications such as cloning, sequencing, and gel electrophoresis.

These purified plasmid samples were sent for third-party sequencing (Plasmidsaurus) to confirm the correct insert sequence and reading frame.

Sequencing results for constructs with different secretion tags are presented in Figures 3–6: DsbA (Figure 3), MalE (Figure 4), TorA (Figure 5), and no-tag (Figure 6). Purified plasmid DNA from each construct was aligned to its *in silico* reference sequence, with matching bases highlighted in red in the alignment images. In the region corresponding to AN-PEP, sequencing consistently revealed large deletions, although the start and end points of these gaps varied between samples. This variability indicates that the deletions were not uniform, suggesting that the AN-PEP coding sequence may be unstable or prone to recombination during cloning.
![Sequencing dsba](https://static.igem.wiki/teams/5785/results/results-dbsa1.webp)

**Figure 3.** Sequencing alignment of the AN-PEP construct with the DsbA secretion tag. Bases matching the *in silico* reference are highlighted in red. Gaps in the sequence indicate deletions within the AN-PEP region, with variable start and end points between samples.

![Sequencing male](https://static.igem.wiki/teams/5785/results/result-male1.webp)

**Figure 4.** Sequencing alignment of the AN-PEP construct with the MalE secretion tag. Bases matching the *in silico* reference are highlighted in red. Deletions are observed in the AN-PEP region, showing inconsistent placement across different colonies.

![Sequencing tora](https://static.igem.wiki/teams/5785/results/result-tora1.webp)

**Figure 5.** Sequencing alignment of the AN-PEP construct with the TorA secretion tag. Red bases indicate matches to the *in silico* reference. Variable deletions are present in the AN-PEP region, with gaps starting and ending at different positions in each sample.

![Sequencing no](https://static.igem.wiki/teams/5785/results/result-no1.webp)

**Figure 6.** Sequencing alignment of the AN-PEP construct without a secretion tag (no tag). Bases in red match the reference sequence. Deletions within the AN-PEP coding region are evident, showing inconsistent placement across samples.

As the sequencing results did not match the expected outcome for the target construct, a second round of colony screening was initiated. To rapidly assess the size and integrity of the plasmid DNA from new colonies, samples were analyzed by gel electrophoresis.
The gel (Figure 7) shows the separation of plasmid fragments from various test colonies, allowing for a size-based comparison to identify potential clones with the correct insert size before proceeding with further sequencing.

![Gel](https://static.igem.wiki/teams/5785/results/results-gel1.webp)

**Figure 7.** Gel electrophoresis of plasmids with varying sizes compared to known controls.
Lanes labeled D, M, T, and N correspond to constructs containing the secretion tags DsbA, MalE, TorA, and no tag, respectively. Lanes marked 1 and 2 represent samples previously sequenced and found to contain deletions. Samples 3, 4, and 5 were isolated from different colonies on their respective plates for comparison.

### Ordered and transformed P61 and P64 into DH5ɑ

The team ordered plasmids containing the transcription units, rehydrated them then transformed them into *E. coli* DH5ɑ. Singular colonies were then cultivated, and from these the plasmids were collected using the Monarch Spin Plasmid Miniprep Kit provided by NEB. Later, optical density analysis was performed (Table 2, supplemental), as well as agarose gel electrophoresis (Figure 8).

The optical density test allowed us to determine the quantity and quality of DNA present in the bacteria. The gel gave the team insight on the success of the Golden Gate assembly. The most visible bands are present at the 3000 bp level, indicating success at including the transcription unit for P61 or P64 into the backbone.

![Gel electrophoresis](https://static.igem.wiki/teams/5785/results/results-gel2-l.webp)

**Figure 8.** Agarose Gel Electrophoresis of P61/P64 transformed DH5-alpha cells with NEB 1kb plus ladder.

![TUs AN-PEP](https://static.igem.wiki/teams/5785/results/results-tus-anpep.webp)

**Figure 9.** Transformed full transcription units (TUs) of AN-PEP cloned into E. coli Nissle.
Plates show colonies expressing AN-PEP constructs with three different secretion tags, alongside two no-tag controls. The plate at the back right serves as a negative control.

![Transformation of Peptide Cap](https://static.igem.wiki/teams/5785/results/results-transformation-nissle.webp)

**Figure 10.** Transformation of AN-PEP Constructs in *E. coli* Nissle.

A single colony from each plate was isolated, streaked out, and preserved for future work, as the remaining colonies were notably small.

![Transformation of Peptide Cap](https://static.igem.wiki/teams/5785/results/results-transformation-dh5.webp)

**Figure 11.** Transformation of Peptide Cap Constructs in *E. coli* DH5α.

Plates were prepared for peptide caps P61 and P64, each with secretion tags (DsbA, MalE, and TorA) and a no-tag control. As shown in Figure 11, sample sites (dots) labeled 1 and 2 indicate colonies selected for mini-prep and downstream testing.

### EZ Gluten Test Results

![Transformation of Peptide Cap](https://static.igem.wiki/teams/5785/results/results-gluten-test.webp)

**Figure 12.** EZ Gluten Test Strips Used to Assess Peptide Cap Binding. Results are shown in the same order as the sample tubes: G1 represents gluten with peptide cap 61, G4 represents gluten with peptide cap 64, and G (far right) is the gluten control (500 ppm). The same layout applies to gliadin samples (D1, D4, and D), with the gliadin control positioned beside the gluten control.

Peptide caps were mixed with equal concentrations of gluten or gliadin and incubated for 15 minutes before testing.
Results were inconclusive, as the gluten control indicated no detectable gluten, while gliadin produced a weak signal similar to the peptide cap samples. It is possible that the low solubility of gluten and gliadin in water contributed to these abnormal readings.

## Supplemental

**Table 1.** Optical density measurements of *E. coli* mini preps to determine quality of purified plasmid preparations

| Secretion Tag Tested | Colony # | OD260 | OD280 | Concentration (ng/µL) | OD260/OD280 Ratio |
| --- | --- | --- | --- | --- | --- |
| No Tag | 3 | 0.0118 | 0.0059 | 11.8 | 1.95 |
| No Tag | 4 | 0.0494 | 0.0267 | 49.4 | 1.84 |
| No Tag | 5 | 0.0824 | 0.0452 | 82.4 | 1.83 |
| DsbA | 3 | 0.1453 | 0.0785 | 145.3 | 1.86 |
| DsbA | 4 | 0.0496 | 0.0272 | 49.6 | 1.85 |
| DsbA | 5 | 0.0555 | 0.0313 | 55.5 | 1.77 |
| TorA | 3 | 0.0491 | 0.0256 | 49.1 | 1.89 |
| TorA | 4 | 0.0349 | 0.0166 | 34.9 | 2.11 |
| TorA | 5 | 0.0512 | 0.0270 | 51.2 | 1.88 |
| MalE | 5 | 0.0528 | 0.0287 | 52.8 | 1.84 |

**Table 2:** Optical density Mmeasurements of *E. coli* mini preps to determine quality of purified preparations from P61 and P64 expressing plasmids

| Secretion Tag | Colony ID Number | OD260 | OD280 | Concentration (ng/µL) | OD260/OD280 Ratio |
| --- | --- | --- | --- | --- | --- |
| MalE | P61 - 1 | 0.3333 | 0.1808 | 333.3 | 1.84 |
| MalE | P61 - 2 | 0.0112 | 0.0049 | 11.2 | 2.22 |
| MalE | P64 - 1 | 0.0097 | 0.0052 | 9.7 | 1.09 |
| MalE | P64 - 2 | 0.0144 | 0.0076 | 14.4 | 1.80 |
| TorA | P61 - 1 | 0.0104 | 0.0056 | 10.4 | 1.89 |
| TorA | P61 - 2 | 0.0064 | 0.0037 | 6.4 | 1.81 |
| TorA | P64 - 1 | 0.0140 | 0.0081 | 14.0 | 1.92 |
| TorA | P64 - 2 | 0.0128 | 0.0070 | 12.8 | 1.91 |
| DsbA | P61 - 1 | 0.0241 | 0.0132 | 24.1 | 1.82 |
| DsbA | P61 - 2 | 0.0204 | 0.0114 | 20.4 | 1.83 |
| DsbA | P61 - H1 | 0.4042 | 0.2189 | 404.2 | 1.85 |
| DsbA | P61 - H2 | 0.3807 | 0.2040 | 380.7 | 1.86 |
| DsbA | P64 - 1 | 0.0454 | 0.0250 | 45.4 | 1.79 |
| DsbA | P64 - 2 | 0.0176 | 0.0091 | 17.6 | 1.82 |
| DsbA | P64 - H1 | 0.0204 | 0.0103 | 20.4 | 2.05 |
| DsbA | P64 - H2 | 0.0224 | 0.0116 | 22.4 | 1.86 |

{% endblock %}