iGEM 2025 — Team dbhs-ca

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## Notebook

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{% block title %}Engineering Success{% endblock %}
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* [Overview](#overview)
* [Cycle 1](#cycle1)
  + [Design](#design1)
  + [Build](#build1)
  + [Test](#test1)
  + [Learn](#learn1)
* [Cycle 2](#cycle2)
  + [Design](#design2)
  + [Build](#build2)
  + [Test](#test2)
  + [Learn](#learn2)
* [Cycle 3](#cycle3)
  + [Design](#design3)
  + [Build](#build3)
  + [Test](#test3)
  + [Learn](#learn3)

## Overview:

As a whole, we went through three rounds of the DBTL cycle. In the first phase, we constructed a plasmid designed for the bacteria *Lactobacillus plantarum* (commonly fermented in probiotics), specifically an spp system that could be induced by sodium acetate. Learning from cycle one, our second cycle was to construct a plasmid for *Escherichia coli, E. coli* for more reliable growth and production. This second plasmid was a pET 21 backbone with our gene of interest, induced and grown in IPTG for production. Our last cycle was the design and testing of our protein of interest itself, where we ran an ELISA assay on our two mutations, comparing it to our original IL-10, to test for their stability. Further details can be found below for all cycles.

## Cycle 1: Constructing a Plasmid

##### Design

We first sought to construct a plasmid that is compatible with the lactobacillus system, settling on the following vector comprised of a mechanism that is similar to what our lactobacillus species, *Lactobacillus plantarum*, processes: a two-component regulatory system homologous to the SppK/SppR system. Our goal was to replace the NucA site (constructed for the study Genome-wide analysis of signal peptide functionality in Lactobacillus plantarum WCFS1 by Mathiesen G, Sveen A, Brurberg MB, Fredriksen L, Axelsson L, Eijsink VG) with our IL-10 mutation variations (original, mutation 1, and mutation 2 IL-10 sequences), created through ThermoMPNN and other modeling softwares that can be found
[here](mutations).

![Addgene Plasmid: locations of the sppK and sppR system](https://static.igem.wiki/teams/5659/engineering/eng1.webp)
![Addgene Plasmid: origin of replication](https://static.igem.wiki/teams/5659/engineering/eng2.webp)

**Figure 1. A simple design of our plasmid sourced from Addgene, detailing the locations of the sppK and sppR system (Left). The origin of replication of the same plasmid is labeled along with restriction enzymes (Right).**
*Source: pLp\_3050sNuc was a gift from Vincent Eijsink & Geir Mathiesen (Addgene plasmid # 122030 ; http://n2t.net/addgene:122030 ; RRID:Addgene\_122030)*

Further plasmid details are provided that can be found in the link
[here](https://www.addgene.org/122030/).
Aspects to note include that it (1) is a spp-based expression vector, (2) has a selectable marker of erythromycin, and (3) has a sppA promoter.

Final sequences were submitted into ApE as a visualization of the expected plasmid produced. We created a total of three constructs that include components (aside from the vector backbone) that include: a 3050 signal peptide designed specifically for gene expression in Lactobacillus plantarum, the IL-10 DNA sequence, a V5 epitope, and an 8x His tag.

![Figure 2](https://static.igem.wiki/teams/5659/engineering/fig2.webp)

**Figure 2. Visualization original IL-10 plasmid’s DNA sequence on ApE (a plasmid editor)**

![Figure 3](https://static.igem.wiki/teams/5659/engineering/fig3.webp)

**Figure 3. Visualization mutation 1 IL-10 plasmid’s DNA sequence on ApE**

![Figure 4](https://static.igem.wiki/teams/5659/engineering/fig4.webp)

**Figure 4. Visualization mutation 2 IL-10 plasmid’s DNA sequence on ApE**

Previous

Next

##### Test

After assembling our plasmids, we attempted to transform them into Lactobacillus plantarum to evaluate whether the constructs could be taken up and maintained by the host. However, the transformation did not yield any viable colonies on selective media, indicating that either the transformation was unsuccessful or the plasmids were not maintained in the cells.

![Figure 5](https://static.igem.wiki/teams/5659/engineering/engfig5.webp)

**Figure 5. Transformation plates showing unsuccessful growth of Lactobacillus plantarum in erythromycin antibiotic.** Multiple selective plates were prepared, each divided into three regions corresponding to the original IL-10, M1-IL-10, and M2-IL-10 constructs. After transformation, no bacterial colonies were observed in any of the regions, indicating that the plasmids were not successfully taken up or maintained by the host.

To investigate the issue, we extracted DNA from the attempted Lactobacillus transformants and analyzed it by agarose gel electrophoresis. The gel showed no visibly apparent bands beyond debris corresponding to the expected plasmid size, suggesting that the plasmids had not been successfully introduced into or propagated within the Lactobacillus cells.

![Figure 6](https://static.igem.wiki/teams/5659/engineering/engfig6.webp)

**Figure 6. Gel electrophoresis image of DNA extracted from attempted Lactobacillus plantarum transformants.** The DNA ladder is visible on the right, but no distinct bands corresponding to the expected plasmid size were observed in the sample lanes. Some debris and background signal were captured on the gel, indicating that the transformation did not yield detectable plasmid DNA.

This outcome highlighted a key limitation in our first cycle: while the plasmid assembly itself appeared correct, the delivery of the construct into Lactobacillus required further optimization, such as refining the transformation protocol or reassessing plasmid compatibility with the host.

##### Build

To assemble our Lactobacillus-compatible plasmids, we first grew up the pLp\_3050sNuc backbone to obtain sufficient plasmid for downstream assembly. The vector was then linearized by restriction enzyme digestion at the NucA site, which we targeted for replacement with our IL-10 constructs.

Rather than PCR-amplifying the inserts, we used pre-ordered synthetic DNA fragments for the three IL-10 variants (original, M1, and M2), each designed to include the 3050 signal peptide for Lactobacillus expression, along with a V5 epitope and an 8×His tag. These synthetic inserts were joined with the digested backbone using enzyme digest and Gibson Assembly, chosen for its efficiency in creating seamless junctions between overlapping sequences.

##### Learn

From this first cycle, we learned that our lack of observable growth and the absence of plasmid bands on the gel left open two possibilities: either the plasmid design or assembly was not fully successful, or the transformation into Lactobacillus plantarum was ineffective. Without clear evidence of plasmid recovery, we could not pinpoint which factor was primarily responsible for the failure.

Given time and budgetary constraints, we determined that additional rounds of troubleshooting in *Lactobacillus* would delay our progress toward demonstrating proof-of-concept. As a result, we strategically pivoted to using the more tractable E. coli system for Cycle 2, which offered well-established protocols, lower costs, and faster turnaround for testing expression and protein stability. This decision allowed us to focus our limited resources on obtaining interpretable experimental data while keeping longer-term Lactobacillus optimization in view for future work.

## Cycle 2: Constructing a Plasmid (Revised)

##### Design

Learning from our first iteration, our second cycle consisted of constructs specific to E. coli, which we were more confident in working with. We utilized a pre-existing expression vector, pET-21(+), as the backbone, specifically for its resistance to antibiotic ampicillin. We have attached an image of the vector below:

![Figure 7](https://static.igem.wiki/teams/5659/engineering/engfig7no2.webp)

**Figure 7. The pET-21(+) vector as detailed by Twist Biosciences.** Components of the plasmid include a lac operator, 6x His tag, T7 promoter, ampicillin resistance gene, and an origin of replication. *Source: Twist Biosciences, Expression Vector Catalog*

Notes about the plasmid: T7 RNA polymerase driven transcription vector for expression in E. coli. The vector, which lacks the ribosome binding site and ATG start codon, is designed for protein expression from translation signals carried by the cloned DNA. Certain features we chose it for include a lac repressor/lac operator, and can be induced by adding lactose or IPTG.

As one might note, one of the key things the plasmid lacks is a ribosome binding site and ATG start codon. Therefore, aside from the components of our plasmid we incorporated in the first cycle (3050 signal peptide, the IL-10 DNA sequence, a V5 epitope, and an 8x His tag), we also added a start codon and ribosomal binding site.

Below is also a sample construct of our designed gene insert. The SD sequence is our ribosomal binding site, the ATG (start codon) included at the start of the signal peptide and IL10 component, and the V5 epitope and 8x His tag at the end. We should also note that the 8x His tag here also corresponds with a 6x His tag that could be found in the vector, but were advised to simply increase the elution concentration in our washing procedures for a cleaner yield.

![Figure 8](https://static.igem.wiki/teams/5659/engineering/fig8.webp)

**Figure 8. Visualization of our construct (starting from ribosomal binding sites to 8x His tag) on ApE;** Excludes the backbone.

##### Build

To test our plasmid, we used a transformation protocol to introduce it into two strains of E. coli, DH5-α and BL21(DE3). Transforming it into DH5-α allowed us to propagate and maintain the plasmid, ensuring stable storage and the creation of a long-term glycerol stock. In contrast, we chose BL21(DE3) as our expression host because it carries a chromosomal copy of the T7 RNA polymerase gene under control of the lacUV5 promoter. This means that when induced with IPTG or lactose, BL21(DE3) cells produce T7 RNA polymerase, which specifically recognizes the T7 promoter in our pET-21(+) plasmid and drives robust transcription of our IL-10 construct. This setup enabled us to pair the cloning efficiency of DH5-α with the high-level protein expression capacity of BL21(DE3), providing an effective workflow for plasmid maintenance and subsequent expression testing.

We used an ampicillin antibiotic to test for bacteria that took up the correct plasmid, verified in the images below.

![Figure 8](https://static.igem.wiki/teams/5659/engineering/engfig9.webp)

**Figure 9. Pictures depict pET M2 IL-10 plasmid in DH5-α (top left), pET M1 IL-10 plasmid in DH5-α (top center), pET original IL-10 plasmid in DH5-α (top right), pET M2 IL-10 plasmid in BL21(DE3) (bottom left), pET original IL10 plasmid in BL21(DE3) (bottom center), and pET M1 IL-10 plasmid in BL21(DE3) (bottom right).**

We next induced expression in verified BL21(DE3) colonies by adding IPTG, preparing the cultures for downstream protein analysis. To do this, individual colonies were first picked and grown in LB medium supplemented with ampicillin until mid-log phase, at which point IPTG was introduced to initiate expression of our IL-10 construct. These induced cultures were subsequently harvested for assays to confirm and evaluate protein production.

##### Test

To assess whether our IL-10 constructs were expressed in E. coli BL21(DE3), we analyzed both total cell lysates and culture supernatants from induced and non-induced cultures using SDS-PAGE followed by anti-His Western blotting. The Coomassie-stained SDS-PAGE gel (Figure 8) shows the overall protein profiles for the samples. Lanes loaded with induced lysates (lanes 3, 5, 7) and supernatants (lanes 9, 11, 13) did not display an obvious new dominant band compared to their non-induced counterparts, suggesting that recombinant IL-10 was not highly abundant in the crude lysate or secreted fraction at detectable levels by Coomassie staining alone.

![Figure 10](https://static.igem.wiki/teams/5659/engineering/engfig10.webp)

**Figure 10. SDS-PAGE analysis of IL-10 expression in BL21(DE3).** Coomassie-stained 4% stacking / 15% resolving SDS-PAGE gel comparing total lysates (lanes 2–7) and culture supernatants (lanes 8–13) from BL21(DE3) expressing wild-type or mutant IL-10, with or without IPTG induction. Lanes 14–15 contain BSA standards. No prominent new band corresponding to IL-10 (~18 kDa) is apparent in induced samples relative to controls.

The Western blot probed with an anti-His antibody (Figure 9), however, revealed a weak but specific signal at approximately the expected size of IL-10 (~18 kDa) in the induced lysate lanes compared to non-induced controls, consistent with low-level expression of the His-tagged IL-10 variants. The absence or faintness of signal in the supernatant lanes suggests that most of the recombinant protein remained intracellular or was produced at levels below the blot’s detection limit in the culture medium. The strong positive bands observed in lanes 14–15 (purified mCherry-His controls) validated the antibody and detection system. These results together indicate that the constructs were expressed but at relatively low levels under the tested conditions, motivating further optimization of induction parameters and potentially the inclusion of secretion-enhancing elements.

![Figure 11](https://static.igem.wiki/teams/5659/engineering/fig11.webp)

**Figure 11. Western blot detection of His-tagged IL-10 in BL21(DE3).** Blot probed with mouse anti-His (1:5000) and HRP-conjugated goat anti-mouse secondary antibody. Weak but distinct bands near ~18 kDa were detected in induced lysate lanes, while supernatant lanes showed minimal signal. Purified mCherry-His in lanes 14–15 served as a positive control.

##### Learn

From this cycle, we learned that while our constructs were successfully introduced and expressed in BL21(DE3), the detected protein levels were relatively low and largely confined to the intracellular fraction. The weak Western blot signal and absence of strong bands in the Coomassie gel suggested that the constructs were functional but sub-optimally expressed under the tested induction conditions.

Although these results highlighted the potential benefit of reconstructing or refining the plasmids (e.g., adding stronger expression or secretion elements), we did not have sufficient time during this cycle to redesign and rebuild new constructs. We kept this consideration in mind as we moved forward. Instead, we prioritized growing an additional batch of induced BL21(DE3) cultures to harvest sufficient protein for downstream testing, focusing on evaluating protein stability using ELISA assays.

## Cycle 3

##### Design

In our third cycle, we shifted our focus from plasmid construction to engineering more stable forms of IL-10 to incorporate into our plasmid (so in a sense it is in lieu with our cycle 2). Our goal was to design IL-10 variants that could retain their structure and function under harsh temperature conditions.

We began by selecting a crystal structure of IL-10 from the Protein Data Bank (PDB ID: 1ILK) as our reference model. This 3-D structural template allowed us to visualize the native fold and the interface of the two subunits that make up the active dimer.

![Figure 12](https://static.igem.wiki/teams/5659/engineering/fig12.webp)

**Figure 12. Crystal structure of human IL-10 homodimer (PDB ID: 1ILK).** The two identical subunits of IL-10 are shown in green and orange ribbon representation. Each monomer contributes to the characteristic intertwined four-helix bundle, forming the biologically active homodimer. This structure, determined by X-ray crystallography at high resolution, served as the starting template for identifying and modeling thermostabilizing point mutations in our design cycle. *Source: RCSB Protein Data Bank, https://www.rcsb.org/structure/1ILK*

To search for stabilizing mutations, we used ThermoMPNN, a machine-learning tool that predicts how point mutations influence a protein’s thermal stability (reported as ΔΔG, where negative values indicate improved stability). We uploaded the IL-10 structure to a Google Colab implementation of ThermoMPNN, toggled off the “include cysteines” setting to avoid disulfide-bond misfolding, and tested the multichain-interface option to ensure we preserved dimer integrity. Out of nearly 7,000 predicted single-amino-acid changes, about 1,000 were predicted to increase stability, becoming the focus of our analysis.

![Figure 13](https://static.igem.wiki/teams/5659/engineering/fig13.webp)

**Figure 13. ThermoMPNN stability heatmap for IL-10 (PDB ID: 1ILK).** Predicted ΔΔG values (kcal/mol) for all single-amino-acid substitutions. Blue indicates stabilizing mutations, while red indicates destabilizing mutations. Clusters of stabilizing sites were notably observed around residues 32-38 and 133-139, guiding candidate selection for thermostable IL-10 variants.

We next used PyMOL to screen the top ~400 mutations, removing those that caused steric clashes or disrupted key hydrogen bonds, salt bridges, or dimer geometry. PyMOL also allowed us to confirm that favorable interactions (such as hydrophobic packing and receptor-binding contacts) were preserved. Because IL-10’s activity depends on binding its receptors (IL-10RA and IL-10RB), we used AlphaFold3 to ensure our candidate mutations did not affect receptor engagement. The modeled complexes showed interface scores (ipTM) similar to wild type, indicating that binding affinity was maintained.

We then combined several top mutations into two final designs and evaluated them with the ESM protein language model, which predicted slightly lower folding free energies (ΔG ≈ 4.26 vs. 4.36 kcal/mol for wild type), suggesting improved thermostability.

Overall, by integrating PyMOL, AlphaFold3, and ESM, we developed two IL-10 variants predicted to resist unfolding at higher temperatures while retaining receptor function. These designs guided the constructs tested in later wet-lab cycles. More about protein design can be found in our Dry Lab subcategory.

##### Build

For the third cycle, we focused on producing the newly designed IL-10 variants in the same bacterial expression system used in the previous cycle. We transformed the plasmids carrying the two selected mutant IL-10 sequences into E. coli BL21(DE3) cells, the standard strain for T7 promoter–driven expression. Successfully transformed colonies were grown in LB medium supplemented with ampicillin to select for plasmid maintenance.

To induce protein production, cultures were grown to mid-log phase and then treated with IPTG, which activates T7 RNA polymerase in BL21(DE3) and drives transcription of the IL-10 constructs. After induction, cells were harvested for downstream analysis and planned stability testing of the mutant proteins. This approach mirrored the procedure established in Cycle 2.

##### Test

To assess the yield and apparent stability of our expressed IL-10 variants, we collected both the cell lysate and the culture supernatant separately from induced BL21(DE3) cultures expressing original IL-10, M1-IL-10, and M2-IL-10. The cells were first disrupted using a continuous-flow cell disruptor to efficiently release soluble intracellular protein, and the lysate was clarified by centrifugation to remove debris.

![Figure 14](https://static.igem.wiki/teams/5659/engineering/fig14.webp)

**Figure 14. Collected supernatant fractions from induced BL21(DE3) cultures expressing IL-10 variants.** Shown are the clarified supernatant fractions from cultures expressing original IL-10 (O-IL-10), M1-IL-10, and M2-IL-10, kept on ice prior to purification. The corresponding cell lysate fractions (not pictured here) were collected in similar 50 mL tubes and resuspended in PBS for parallel purification and downstream ELISA analysis.

All three constructs supernatant and lysate were then subjected to nickel-affinity purification using the NEB Ni-NTA bead protocol. The His-tagged IL-10 proteins bound to the resin, were washed to remove non-specific contaminants, and eluted with an imidazole-containing buffer optimized for recovery. Due to our plasmid construct containing greater amounts of His tags, we eluted with a solution containing greater imidazole than the protocol recommended. To examine thermal stability, the purified eluates were split into two aliquots and incubated at 37 °C and 55 °C for 10 minutes before proceeding to quantification.

Following this treatment, all samples were analyzed using a commercial human IL-10 ELISA kit to quantify protein recovery and to evaluate the relative thermal stability of the wild-type and mutant IL-10 constructs. This approach provided a quantitative comparison across both expression localization (lysate vs. supernatant) and construct performance under mild heat stress. By comparing ELISA signals across these conditions, we were able to begin assessing whether our engineered mutations improved IL-10 recovery and stability.

![Figure 15](https://static.igem.wiki/teams/5659/engineering/fig15.webp)

**Figure 15. IL-10 ELISA plate showing color development prior to (left) and after addition (right) of stop solution.** The standard curve wells are the furthest column on the left, showing decreasing concentrations of IL-10. The remaining wells contain the lysate and supernatant samples from original IL-10, M1-IL-10, and M2-IL-10 after purification and thermal treatment for two batches in triplicates. The darker the color, the more present the protein.

![Figure 16](https://static.igem.wiki/teams/5659/engineering/fig16.webp)

**Figure 16. Raw absorbance data from IL-10 ELISA.** Image of the Gen5 plate-reader output showing the optical density (OD\_{450}) values for the IL-10 ELISA plate. These raw OD\_{450} values were used to generate the standard curve and calculate relative IL-10 concentrations for downstream analysis.

##### Learn

This cycle was successful, as we were able to express, purify, and detect our IL-10 variants using ELISA after nickel-affinity purification. Importantly, the workflow (beginning with separate collection of lysate and supernatant, followed by thermal treatment and quantitative ELISA) validated our ability to not only recover the protein but also assess its stability under different conditions. The quantitative outcomes from the ELISA are presented in the
<results> tab, providing key insights into the relative performance of the wild-type and mutant IL-10 constructs.

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{% set boxes = [
{
'id':'box-one',
'title':'Section 1',
'sections': [
{
'slug':'ecoli',
'title':'E.Coli Culture',
'text':'

### Escherichia coli Culture ( E. coli )

##### Materials

* Media: LB (routine), SOC (post-transformation recovery), TB/Terrific Broth (high density).
* Antibiotics (working): Amp 100 µg/mL; Kan 50 µg/mL; Cm 25 µg/mL; Spec 50 µg/mL.
* Plates: LB-agar + appropriate antibiotic.
* Consumables: Sterile loops/tips, 14/50 mL tubes, baffled flasks (5x culture volume).
* Equipment: 37 °C incubator, shaking incubator (200-250 rpm, 25 mm orbit), spectrophotometer (OD600).

##### Media (1 L)

* LB: 10 g tryptone, 5 g yeast extract, 10 g NaCl; dHO1 L; autoclave.
* LB-Agar: LB + 15 g agar; autoclave, cool to 55 °C, add antibiotic, pour plates.
* SOC (ready-made preferred) for post-transformation.
* TB: 12 g tryptone, 24 g yeast extract, 4 mL glycerol; dHO900 mL; autoclave. After cooling add 100 mL 10x TB salts (0.17 M KHPO, 0.72 M KHPO).

##### Protocols

### Day 0: Streak for Single Colonies

1. Warm selective plate (room temp, lid down).
2. Flame loop touch glycerol stock/colony streak for isolation (quadrant streak).
3. Invert and incubate 12-16 h at 37 °C (or 24 h if slow).

### Day 1: Starter Culture (Overnight)

1. Pick one well-isolated colony inoculate 3-5 mL LB + antibiotic in a 14 mL tube.
2. Incubate 37 °C, 220-250 rpm, ~12-16 h (loosely capped for aeration).

* Expression strains for toxic proteins: consider 30 °C overnight to reduce stress.

### Day 2: Main Culture

1. Inoculate baffled flask with 1:100 from starter (e.g., 5 mL into 500 mL LB/TB + antibiotic).
2. Incubate 37 °C, 200-250 rpm until desired OD600:
   * Mid-log: 0.4-0.6 (ideal for induction/transformation-related assays).
   * Late-log: 0.8-1.0 (yield vs. stress trade-off).
3. Proceed to the downstream step (induction, plasmid prep, harvest, etc.).

### Protein Expression (BL21(DE3))

* At OD600 0.5-0.7, add IPTG 0.1-1 mM (typical 0.5 mM).
* Express 2-4 h at 30-37 °C (fast, may form inclusion bodies) or 16-20 h at 16-20 °C (solubility).
* Harvest: 4,000-6,000xg, 10 min, 4 °C; proceed to lysis.

'
},
{
'slug':'bacterialtransformation',
'title':'Bacterial Transformation',
'text':'

**Source:** [Addgene protocol](https://www.addgene.org/protocols/bacterial-transformation/)

##### Equipment

* Shaking incubator at 37 °C
* Water bath at 42 °C
* Ice bucket filled with ice
* Microcentrifuge tubes
* Sterile spreading device

##### Reagents

* LB agar plate
* LB or SOC media
* Competent cells
* DNA for transformation

##### Protocol

1. Take competent cells out of -80 °C and thaw on ice (approximately 20-30 mins).
2. Remove agar plates (containing the appropriate antibiotic) from storage at 4 °C and let them warm up to room temperature and then (optional) incubate in a 37 °C incubator.
3. Mix 1-5 µL of DNA (usually 10 pg-100 ng) into 20-50 µL of competent cells in a microcentrifuge or falcon tube. Gently mix by flicking the bottom of the tube with your finger a few times.
   **Note:** Transformation efficiencies will be approximately 10-fold lower for ligation of inserts to vectors than for an intact control plasmid.
4. Incubate the competent cell/DNA mixture on ice for 20-30 mins.
5. Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42 °C water bath for 30-60 secs (45 secs is usually ideal, but this varies depending on the competent cells you are using).
6. Put the tubes back on ice for 2 min.
7. Add 250-1,000 µL LB or SOC media (without antibiotic) to the bacteria and grow in a 37 °C shaking incubator for 45 min.
   **Note:** This outgrowth step allows the bacteria time to generate the antibiotic resistance proteins encoded in the plasmid backbone so that they will be able to grow once plated on the antibiotic containing agar plate. This step is not critical for Ampicillin resistance but is much more important for other antibiotic resistances.
8. Plate some or all of the transformation onto a 10 cm LB agar plate containing the appropriate antibiotic.
9. Incubate plates at 37 °C overnight.

![Transformation procedure diagram](https://static.igem.wiki/teams/5659/experiments/unnamed.webp)

Transformation procedure diagram

'},
{'slug':'sdspage','title':'SDS Page','text':'

##### Summary

20uL of sample was mixed with 6.6uL of 4X LDS-PAGE loading (Cat# NP0008) buffer containing
10% v/v beta-mercaptoethanol. Samples were boiled for 10min at 95C and then spun in a
centrifuge at 20,000xg for 30seconds. The entire 26.6uL volume was loaded into wells of a
self-cast 4% stacking/10% resolving gel (1.5mm thickness). The gel was run at 200V for ~40min
in a tris-glycine buffer according to Bio-Rad instructions for SDS-PAGE (Laemmli) buffer system.

**Source** : Biorad Mini-PROTEAN 3 Cell Protocol

##### Protocol

### Gel Cassette Preparation

1. Ensure glass plates, casting frames, and stands are clean and dry.
2. Place Casting Frame upright with cams open.
3. Insert Spacer Plate (label side up) with a Short Plate in front.
4. Slide plates into frame with short plate facing forward; lock cams to seal.
5. Place the cassette on the casting stand, making sure the gasket seals.
6. Repeat for second gel if needed.

### Gel Casting (Hand-Cast)

Discontinuous Gel (standard for SDS-PAGE)

1. Insert comb, mark gel at 1 cm below comb teeth, remove comb.
2. Prepare resolving gel mix (all but APS/TEMED); degas 15 min.
3. Add APS/TEMED, pour to mark, overlay with water or t-amyl alcohol, polymerize 45-60
   min, rinse.
4. (Optional overnight storage: overlay with 5 mL 1x resolving gel buffer.)
5. Prepare stacking gel mix, degas 15 min.
6. Dry area above resolving gel, add APS/TEMED, pour to top of short plate.
7. Insert comb carefully, polymerize 30-45 min, remove comb, rinse wells.

### Continuous Gel

1. Prepare and degas monomer solution.
2. Add APS/TEMED, pour to top of short plate.
3. Insert comb, polymerize 45-60 min, remove comb, rinse wells.

### Ready-Gel Option

1. Remove precast gel from pouch, take out comb, rinse wells.
2. Cut a dotted line at the bottom and peel sealing tape to expose the gel edge.

### Module Assembly

1. Remove gel sandwiches from the casting stand; unlock from frames.
2. Insert into electrode assembly with short plates facing inward.
3. Close clamps to seal; lower inner chamber into tank.
4. Add ~125 mL running buffer to inner chamber and ~200 mL to outer tank.

### Sample Prep & Loading

1. Mix 20 µL protein sample with 6.6 µL 4x LDS-PAGE buffer + 10% -mercaptoethanol.
2. Boil 10 min at 95 °C, spin 20,000xg for 30 s.
3. Load full volume (~26.6 µL) into wells with gel-loading tips; load slowly to avoid
   puncturing wells.

### Running the Gel

1. Place lid on tank, align color-coded leads.
2. Run at 200 V constant for ~35-40 min in Tris-glycine (Laemmli) buffer.

### Gel Removal & Cleanup

1. Turn off power, disconnect leads, remove lid.
2. Pour off the buffer before opening clamps.
3. Separate sandwich plates; loosen gel with wedge tool or razor and float into fixative or
   transfer buffer.
4. Rinse tank, frames, and electrode assembly with distilled water after use.

### Notes:

* Ensure proper gasket seal to prevent leaks.
* Avoid leaving alcohol overlay on gel for > 1 h to prevent dehydration.
* Refer to the Bio-Rad Mini-PROTEAN 3 Cell User Guide for full details.

'},
{'slug':'westernblotting','title':'Western Blot','text':'

##### Summary

The gel was transferred onto a 0.2um nitrocellulose membrane according to Bio-Rad
instructions using the Mini-Transblot cell. 100V was applied for 60min in tris-glycine buffer
containing 20% v/v MeOH. The blot was blocked with 1% casein in 1x PBS followed by probing
with mouse anti-His at 0.1 ug/mL in 1% casein (Genscript A00186) followed by HRP-conjugated
goat anti-Mouse (subclass specific) (Jackson Cat 115-035-164) at 0.16 ug/mL in 1% casein.
The blot was then developed using DAB (Sigma Cat D-5905)
For further details, please refer to the source.

**Source:** Biorad Mini Trans-Blot Electrophoretic Transfer Cell Protocol

##### Protocol

### Prep & Safety

1. Clean before use: Wash tank, cassettes, fiber pads with mild lab cleaner; rinse DI water.
2. Safety interlock: Power off before removing lid. Max ratings (instrument level): 400
   VDC, 500 W, 40 °C.
3. Cooling: Store blue cooling unit at 20 °C; insert frozen for every run. Use a stir bar in the
   tank (mix fast) to keep temperature/ions uniform.

### Buffers & Materials

1. Transfer buffer (cold, 4 °C): Common options (see Section 3.3 of manual):
   * Tris-glycine (25 mM Tris, 192 mM glycine 20% MeOH, pH 8.3) - standard
     SDS-PAGE transfers.
   * 48 mM Tris/39 mM glycine 20% MeOH, pH 9.2 - alternative.
   * 10 mM NaHCO/3 mM NaCO 20% MeOH, pH 9.9 - useful for basic proteins.
   * Do not adjust pH unless specified; use analytical-grade methanol; do not reuse
     buffer.
2. Membrane:
   * PVDF - prewet 15 s in 100% MeOH, then equilibrate in transfer buffer.
   * Nitrocellulose - equilibrate in transfer buffer (binding often improved with 20%
     MeOH).
3. Cut to size: Membrane, filter papers, and fiber pads trimmed to gel; pre-soak 15-20 min
   in transfer buffer (also equilibrate gel).

### Cassette Sandwich (Cathode Anode)

1. On cassette base (gray side down): Fiber pad Filter paper Gel (no bubbles)
   Membrane (no bubbles) Filter paper Fiber pad.
2. Gently roll out all bubbles (glass rod/pipette).
3. Close and latch cassette without shifting layers.
4. For acidic transfers (Section 2.3): swap gel/membrane order so membrane sits on the
   cathode side (proteins move toward ).

### Tank Setup & Run

1. Place cassettes into electrode module (color-coded orientation), add frozen blue cooling
   unit.
2. Insert module into tank; fill to blotting mark with cold transfer buffer (950 mL with
   cooling unit). Add stir bar and mix fast.
3. Attach lid (safety interlock), connect leads.
4. Typical conditions (from Table 3.1):
   * Overnight/standard: low voltage (e.g., 30 V constant).
   * High-intensity wet transfer (mini gels): ~100 V, ~1 h (watch heating).
   * Always keep the unit cold (cooling unit + stirring).
5. On completion: power off, disconnect, remove cassette, recover membrane, rinse briefly
   (e.g., TBST/PBS), proceed to blocking.

### Notes:

* Pre-equilibrate gels (15-20 min) to remove salts and prevent shrinkage.
* Orientation check: Gel toward cathode (), membrane toward anode (+) (except acidic
  mode).
* High MW proteins: consider less MeOH, add 0.05-0.1% SDS, longer time, basic buffer
  (pH 9.5-10); PVDF preferred.
* Low MW proteins: 0.2 µm nitrocellulose or PVDF; shorten time to avoid blow-through.
* Do not adjust buffer pH unless directed; changes can spike current/heat.
* Bubbles/contact: Most common cause of swirls/missing bands-always roll out bubbles
  and use fresh pads/papers

'},
{
'slug':'proteinpurification',
'title':'Protein Purification (Nickel Beads)',
'text':'

**Source:**
[Invitrogen Human IL-10 ELISA Kit - Product Information Sheet](https://documents.thermofisher.com/TFS-Assets/LSG/manuals/MAN0003760_KHC0101_02_01C_Human_IL10_ELISA_PI.pdf)

![Ni-NTA purification buffers and overview](https://static.igem.wiki/teams/5659/experiments/proteinpurification/purification1.webp)

Ni-NTA purification buffers and overview

##### Protocol

*Note:* Binding capacity depends on target and conditions; as a planning estimate, ~50 µL bead slurry often yields on the order of tens of micrograms of purified His-tagged protein. Optimize for your construct.

### Buffer preparation

* 2x IMAC Buffer (0.04 M sodium phosphate, 0.6 M NaCl, pH 7.4)
* 2 M Imidazole (pH 7.4)
* Prepare native binding, wash, and elution buffers from concentrates according to the resin/system used.

### Bead equilibration

1. Resuspend bead slurry thoroughly.
2. Dispense 50 µL slurry to a 1.5 mL tube; add 200 µL binding buffer, mix, place on magnet, discard supernatant.

### Binding

1. Add 1.0 mL clarified lysate to equilibrated beads.
2. Incubate 30 min at room temperature with mixing or ~850 rpm shaking.
3. Place on magnet; keep supernatant as flow-through if needed.

### Wash

1. Add 500 µL wash buffer, resuspend; pellet on magnet; remove supernatant.
2. Repeat wash 3x total; remove residual buffer after final wash.

### Elution

1. Add 100 µL elution buffer; shake ~2 min at ~850 rpm.
2. Place on magnet and collect eluate. Optional: repeat and pool.

![Equilibration, binding, wash, and elution steps](https://static.igem.wiki/teams/5659/experiments/proteinpurification/purification2.webp)

Equilibration, binding, wash, and elution steps

'
},
{
'slug':'elisa',
'title':'ELISA Assay',
'text':'

**Source:**
[Invitrogen Human IL-10 ELISA Kit (KHC0101) - Product Information Sheet](https://documents.thermofisher.com/TFS-Assets/LSG/manuals/MAN0003760_KHC0101_02_01C_Human_IL10_ELISA_PI.pdf)

**Kit components**

![ELISA figure 1](https://static.igem.wiki/teams/5659/experiments/elisa/elisa1.webp)

ELISA figure 1

Required materials not supplied

* Distilled or deionized water
* Calibrated adjustable precision pipettes and tubes for dilutions
* Beakers, flasks, and cylinders for reagent preparation
* Microtiter plate reader (450 nm; optional 550 nm reference)
* Plate washer

##### Protocol

### Buffer preparation

* Dilute 16 mL Wash Buffer Concentrate (25x) with 384 mL water to make 1x Wash Buffer.
* Store concentrate and 1x buffer at 2-8 °C; use diluted buffer within 14 days.

### Dilute standards

Note: This assay has been calibrated against the International Standard preparation (92/516) for human IL-10 (NIBSC, Hertfordshire, UK, EN6 3QG).

1. Reconstitute Hu IL-10 Standard to 5,000 pg/mL with Standard Dilution Buffer. Refer to the standard vial label for instructions. Swirl or mix gently and allow the contents to sit for 10 minutes to ensure complete reconstitution. Label as 5,000 pg/mL human IL-10. Use the standard within 1 hour of reconstitution.
2. Add 50 µL Reconstituted Standard to one tube containing 450 µL Standard Diluent Buffer and mix. Label as 500 pg/mL human IL-10.
3. Add 200 µL Standard Diluent Buffer to each of 7 tubes labeled as follows: 250, 125, 62.5, 31.2 ,15.6, 7.8, and 0 pg/mL human IL-10.
4. Make serial dilutions of the standard as shown in the following dilution diagram. Mix thoroughly between steps.
5. Discard any remaining reconstituted standard. Return the Standard Diluent Buffer to the refrigerator.

![ELISA figure 2](https://static.igem.wiki/teams/5659/experiments/elisa/elisa2.webp)

ELISA figure 2

### Prepare 1x Streptavidin-HRP

Note: Prepare 1X Streptavidin-HRP within 15 minutes of usage. The Streptavidin-HRP (100X) is in 50% glycerol, which is viscous. To ensure accurate dilution:

1. For each 8-well strip used in the assay, pipet 10 µL Streptavidin-HRP (100X) solution, wipe the pipette tip with clean absorbent paper to remove any excess solution, and dispense the solution into a tube containing 1 mL of Streptavidin-HRP Diluent. Mix thoroughly.
2. Return the unused Streptavidin-HRP (100X) solution to the refrigerator.

### Perform Assay (Total assay time: 5 hours)

Note: Allow all components to reach room temperature before use. Mix all liquid reagents prior to use. Determine the number of 8-well strips required for the assay. Insert the strips in the frames for use. Re-bag any unused strips and frames, and store at 2°C to 8°C for future use.

![Key for ELISA figures](https://static.igem.wiki/teams/5659/experiments/elisa/elisakey.webp)

Key for ELISA figures

### 1) Bind Antigen

1. Add 50 µL of Incubation Buffer to wells for serum or plasma samples, and standards; or 50 µL of Standard Diluent Buffer to the wells for cell culture samples. Leave the wells for chromogen blanks empty.
2. Add 50 µL of standards, controls, or samples (see Pre-dilute samples) to the appropriate wells. Leave the wells for chromogen blanks empty.
3. Tap the side of the plate to mix. Cover the plate with a plate cover and incubate for 2 hours at room temperature.
4. Thoroughly aspirate the solution and wash wells 4 times with 1X Wash Buffer.

### 2) Add Biotin Conjugate

1. Add 100 µL Human IL-10 Biotin Conjugate to each well (except blanks). Incubate 2 h at room temperature. Wash 4x.

### 3) Add Streptavidin-HRP

1. Add 100 µL of 1x Streptavidin-HRP to each well (except blanks). Incubate 30 min at room temperature. Wash 4x.

### 4) Add TMB

1. Add 100 µL TMB to each well. Incubate 30 min at room temperature protected from light; solution turns blue.

### 5) Add Stop Solution

1. Add 100 µL Stop Solution to each well; tap to mix. Solution turns yellow.

![ELISA figure 3](https://static.igem.wiki/teams/5659/experiments/elisa/elisa3.webp)

ELISA figure 3

![ELISA figure 4](https://static.igem.wiki/teams/5659/experiments/elisa/elisa4.webp)

ELISA figure 4

![ELISA figure 5](https://static.igem.wiki/teams/5659/experiments/elisa/elisa5.webp)

ELISA figure 5

![ELISA figure 6](https://static.igem.wiki/teams/5659/experiments/elisa/elisa6.webp)

ELISA figure 6

![ELISA figure 7](https://static.igem.wiki/teams/5659/experiments/elisa/elisa7.webp)

ELISA figure 7

##### Read plate and calculate

* Read absorbance at 450 nm within 2 h (optional 550 nm reference).
* Use 4-parameter logistic (4-PL) curve fit; subtract background if available.
* Multiply sample values by dilution factor.

##### Performance characteristics

* Range: 0-500 pg/mL
* Sensitivity: less than 1 pg/mL
* Precision: typical intra-assay CV 3-5%; inter-assay CV 3-6%

'
},
{
'slug':'gibson',
'title':'Gibson Assembly',
'text':'

**Source:** NEB Gibson Assembly Protocol
**Kit components**

![](https://static.igem.wiki/teams/5659/experiments/gibson1.webp)

*Required materials not supplied*

* Recommended PCR polymerases
  + Q5 High-Fidelity DNA Polymerase
  + Q5 Hot Start High-Fidelity DNA Polymerase
  + Q5 Hot Start High-Fidelity 2X Master Mix
* LB plates with appropriate antibiotic(s)

##### Procedure

1. Set up the following reaction on ice:

![](https://static.igem.wiki/teams/5659/experiments/gibson2.webp)

*Optimized cloning efficiency is 50-100 ng of vector with 2-3 fold molar excess of each insert. Use 5-fold molar excess of inserts less than 200 bp. For 4-6 fragment assemblies, use 1:1 insert:vector molar ratio. Total volume of unpurified PCR fragments should not exceed 20% of the assembly reaction.*

2. Incubate in a thermocycler at 50 °C for 15 min (2-3 fragments) or 60 min (4-6 fragments). Store on ice or at 20 °C for transformation.
3. Transform competent E. coli with 2 µL of assembly reaction.

'
},
{'slug':'enzymedigestion','title':'Enzyme Digestion','text':'

### NEB enzyme digestion (Ndel & EcoRI double digest)

**Source:** NEB product pages and general guidelines for Restriction Endonucleases (e.g., NdeI, EcoRI/EcoRI-HF, CutSmart Buffer)

##### Kit components (typical NEB catalog)

* NdeI
* EcoRI or EcoRI-HF
* CutSmart Buffer (10x)
* Gel Loading Dye (optional

Compatibility note: NdeI and EcoRI/EcoRI-HF both show 100% activity in 1x CutSmart Buffer at 37 °C, enabling a one-tube double digest.

##### Required materials not supplied

* DNA (plasmid or PCR product)
* Nuclease-free water
* Optional: BSA (already included in CutSmart), DNA ladder, agarose gel reagents
* Optional cleanup: spin column or magnetic-bead DNA purification kit

##### Notes

### Unit definition

For restriction enzymes, 1 Unit (U) is the amount of enzyme required to completely digest 1 µg of DNA in 1 hour at the recommended temperature in a 50 µL reaction.

### General assay/handling conditions

* Recommended working temperature: 37 °C for both NdeI and EcoRI/EcoRI-HF.
* Keep total glycerol 5% v/v in the final reaction (enzyme stocks are ~50% glycerol).
* Use 1x CutSmart as the buffer (includes BSA).
* Typical enzyme usage: 5-10 U per enzyme per µg DNA (do not exceed 10% of reaction volume with enzyme).

### Methylation & star activity

* Routine Dam/Dcm methylation does not block EcoRI or NdeI.
* To minimize star activity: keep the correct buffer, avoid prolonged incubations, high glycerol, or very low ionic strength; do not over-digest.

##### Protocol

1. **Thaw & mix.** Thaw 10x CutSmart Buffer and DNA on ice. Keep enzymes on a cold rack. Briefly vortex buffer and water; do not vortex enzymes (flick or pipette gently)
2. **Assemble on ice in this order:**

   a) Water b) 10x CutSmart c) DNA d) NdeI e) EcoRI/EcoRI-HF.
   Mix by gentle pipetting; quick-spin to collect contents.
3. **Incubate.** Place at 37 °C for 30-60 min. For difficult templates or >1 µg DNA, extend to 60-90 min (avoid very long incubations to minimize star activity)
4. **Check completion (optional).** Run 2-5 µL on an agarose gel to verify the expected digestion pattern.
5. **Heat-inactivate or clean up.**

   * EcoRI-HF/EcoRI and NdeI are generally heat-inactivatable at 65 °C for ~20 min; however, because double-digests combine enzymes, the most reliable approach is column or bead cleanup to remove enzymes and buffer prior to ligation or downstream steps
6. **Proceed to ligation or analysis.** For cloning, purify the digested insert/vector (gel extraction if you need size selection), then set up ligation.

'}
]
},
{
'id':'box-two',
'title':'Section 2',
'sections': [
{'slug':'glycerolstock','title':'Glycerol Stock','text':'

### Glycerol Stock

**Source:** [Addgene Protocols Creating Bacterial Glycerol Stocks for Long-term Storage of
Plasmids](https://www.addgene.org/protocols/create-glycerol-stock/)

*\*Follow the steps for [Inoculating an Overnight Liquid Culture](https://www.addgene.org/protocols/inoculate-bacterial-culture/).*

##### Materials

* Glycerol
* Sterile Loop
* Overnight Bacterial Culture
* Screwtop or Cyrocrovial Tube

##### Protocol

1. After you have bacterial growth, add 500 µL of the overnight culture to 500 µL of 50%
   glycerol in a 2 mL screw top tube or cryovial and gently mix.
2. Freeze the glycerol stock tube at -80°C. The stock is now stable for years, as long as it is
   kept at -80°C. Subsequent freeze and thaw cycles reduce shelf life.
3. To recover bacteria from your glycerol stock, open the tube and use a sterile loop,
   toothpick or pipette tip to scrape some of the frozen bacteria off of the top. Do not let
   the glycerol stock unthaw! Streak the bacteria onto an LB agar plate.
4. Grow your bacteria overnight at the appropriate temperature. Growth conditions,
   including copy number and growth temperature, can be found on your plasmid\'s
   information page. The next day you will be able to start an overnight culture for plasmid
   DNA prep the following day

<https://www.addgene.org/protocols/create-glycerol-stock/>
<https://www.addgene.org/protocols/create-glycerol-stock/>
<https://www.addgene.org/protocols/inoculate-bacterial-culture/>

'},
{
'slug':'pcr',
'title':'PCR',
'text':'

### Polymerase Chain Reaction (PCR)

**Source:**
[Q5® High-Fidelity DNA Polymerase](https://www.neb.com/en-us/products/m0491-q5-high-fidelity-dna-polymerase)

##### Kit components

### Required materials not supplied

* Deoxynucleotide (dNTP)
* Nuclease-free water

##### Notes

### Unit Definition

* One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 74°C.

### Unit Assay Conditions

* 25 mM TAPS-HCl (pH 9.3 @ 25°C), 50 mM KCl, 2 mM MgCl2, 1 mM β-mercaptoethanol, 200 µM dNTPs including [3H]-dTTP and 15 nM primed M13 DNA.

##### Protocol

Assemble all reaction components on ice. Each component should be gently mixed before adding to the reaction in a sterile thin-walled PCR tube. The Q5 High-Fidelity DNA Polymerase may be diluted in 1X Q5 Reaction Buffer just prior to use to reduce pipetting errors. The entire reaction should be mixed again to ensure a homogeneous, consistent mixture. Collect all liquid to the bottom of the tube with a quick centrifuge spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.

Quickly transfer the reactions to a thermocycler preheated to the denaturation temperature (98°C) and begin thermocycling.

![PCR overview and setup](https://static.igem.wiki/teams/5659/experiments/pcr1.webp)

PCR overview and setup

<https://www.neb.com/en-us/products/m0491-q5-high-fidelity-dna-polymerase>

##### Primer Design Guidelines:

### 1. Template

Use of high-quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template for a 50 µL reaction are as follows:

![Thermocycling recommendations](https://static.igem.wiki/teams/5659/experiments/pcr2.webp)

Thermocycling recommendations

### 2. Primers

Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%. Computer programs such as Primer3 can be used to design or analyze primers. The best results are typically seen when using each primer at a final concentration of 0.5 µM in the reaction. However, amplification of certain long, complex DNA targets (≥ 5 kb) may benefit from using a lower primer concentration (~0.2 to 0.3 µM).

### 3. Mg2+ and additives

Mg2+ concentration of 2.0 mM is optimal for most PCR products generated with Q5 High-Fidelity DNA Polymerase. When used at a final concentration of 1X, the Q5 Reaction Buffer provides the optimal Mg2+ concentration.

### 4. Deoxynucleotides

The final concentration of dNTPs is typically 200 µM of each deoxynucleotide. Q5 High-Fidelity DNA Polymerase cannot incorporate dUTP and is not recommended for use with uracil-containing primers or templates. Should uracil-containing primers or templates be used, we recommend Q5U® Hot Start High-Fidelity DNA Polymerase.

### 5. Q5 High-Fidelity DNA Polymerase concentration

We generally recommend using Q5 High-Fidelity DNA Polymerase at a final concentration of 20 units/mL (1.0 unit/50 µL reaction). However, the optimal concentration of Q5 Hot Start High-Fidelity DNA Polymerase may vary from 10–60 units/mL (0.5–3 units/50 µL reaction) depending on amplicon length and difficulty.

### 6. Buffers

The 5X Q5 Reaction Buffer provided with the enzyme is recommended as the first-choice buffer for robust, high-fidelity amplification. For difficult amplicons, such as GC-rich templates or those with secondary structure, the addition of the Q5 High GC Enhancer can improve reaction performance. The 5X Q5 Reaction Buffer is detergent-free and contains 2.0 mM Mg2+ at the final (1X) concentration.

### 7. Denaturation

An initial denaturation of 30 seconds at 98°C is sufficient for most amplicons from pure DNA templates. Longer denaturation times can be used (up to 3 minutes) for templates that require it.

### 8. Annealing

Optimal annealing temperatures for Q5 High-Fidelity DNA Polymerase tend to be higher than for other PCR polymerases. The NEB Tm Calculator should be used to determine the annealing temperature when using this enzyme. Typically, use a 10–30 second annealing step at the recommended Ta. A temperature gradient can also be used to optimize the annealing temperature for each primer pair.

### 9. Extension

The recommended extension temperature is 72°C. Extension times are generally 20–30 seconds per kb for complex, genomic samples, but can be reduced to 10 seconds per kb for simple templates (plasmid, *E. coli*, etc.) or complex templates < 1 kb. Extension time can be increased to 40 seconds per kb for cDNA or long, complex templates, if necessary.

### 10. Cycle number

Generally, 25–35 cycles yield sufficient product. For genomic amplicons, 30–35 cycles are recommended.

### 11. 2-step PCR

When primers with annealing temperatures ≥ 72°C are used, a 2-step thermocycling protocol (combining annealing and extension into one step) is possible.

### 12. Amplification of long products

When amplifying products > 6 kb, it is often helpful to increase the extension time to 40–50 seconds/kb.

![Primer design and template guidance](https://static.igem.wiki/teams/5659/experiments/pcr3.webp)

Primer design and template guidance

### 13. PCR product

The PCR products generated using Q5 High-Fidelity DNA Polymerase have blunt ends. If cloning is the next step, then blunt-end cloning is recommended. If T/A-cloning is preferred, the DNA should be purified prior to A-addition, as Q5 High-Fidelity DNA Polymerase will degrade any overhangs generated. The Monarch® Spin PCR & DNA Cleanup Kit (5 µg) (NEB #T1130) is recommended as an efficient method for purification and concentration up to 5 µg of high-quality, double-stranded and single-stranded DNA.

Addition of an untemplated -dA can be done with Taq DNA Polymerase.

![PCR product workflow](https://static.igem.wiki/teams/5659/experiments/pcr4.webp)

PCR product workflow

'
},
{'slug':'plasmidextraction','title':'Plasmid Extraction','text':'

**Source:** [Addgene protocol](https://www.addgene.org/protocols/bacterial-transformation/)

## Equipment

* Shaking incubator at 37 °C
* Water bath at 42 °C
* Ice bucket filled with ice
* Microcentrifuge tubes
* Sterile spreading device

##### Reagents

* LB agar plate
* LB or SOC media
* Competent cells
* DNA for transformation

##### Protocol

Buffer Preparation

* Add supplied Monarch RNase A to Monarch Buffer B1 to 100 µg/mL; store at 4 °C.
* Add isopropanol to Buffer BZ (0.43 volumes per volume of BZ).
* Add ethanol to Buffer WZ (4 volumes per volume of WZ).

*Keep buffer bottles tightly closed.*

Plasmid Miniprep Protocol (Centrifugation)

1. Pellet 1-5 mL culture (not to exceed 15 OD units) for 30 s. Discard supernatant.
2. Resuspend pellet in 200 µL Buffer B1.
3. Add 200 µL Buffer B2, invert 5-6x, incubate 1 min. Do not vortex.
4. Add 400 µL Buffer B3, invert to neutralize. Incubate 2 min.
5. Centrifuge 2-5 min.
6. Transfer supernatant to Spin Column S2D; spin 1 min. Discard flow-through.
7. Add 200 µL Buffer BZ; spin 1 min.
8. Wash with 400 µL Buffer WZ; spin 1 min.
9. Transfer column to clean tube.
10. Add 30 µL Buffer EY to matrix center; wait 1 min; spin 1 min to elute DNA.

![](https://static.igem.wiki/teams/5659/experiments/plasmidextraction/pe2.webp)
'},
{'slug':'lactobacillus-culture','title':'Lactobacillus Culture','text':'

### Lactobacillus Culture

**Source:** [Difco Lactobacilli Agar](https://www.bd.com/resource.aspx?IDX=14647)

##### Materials

* **For Agar:**
  + Dehydrated MRS Agar powder: 48 g in 1 L purified water (per manual).
  + Alternative version: 70 g/L depending on product revision.
* **For Broth:**
  + Same MRS formula, omit agar (38 g/L per manual).
* Purified water
* Autoclave & sterilization equipment
* Sterile glassware (flasks, bottles, pipettes)
* Sterile Petri dishes (for agar)
* Inoculating tools (loops, needles)
* Incubator set to appropriate temperature (30-37 °C)
* Control (reference) culture strain for performance testing

##### Protocol

### Agar & Broth Preparation

1. **Dissolve medium.**
   * Weigh the dehydrated powder (agar or broth) and suspend in 1 L purified water.
   * Heat gently with stirring, and boil 1-3 min (per version) to completely dissolve.
2. **Sterilize.** Autoclave at 121 °C for 15 minutes.
3. **Quality check / controls.** After sterilization, test using control cultures to verify expected Lactobacillus growth.

### Using the Media: Inoculation & Culture

#### For Plates (Solid Agar)

1. Pour ~15-20 mL of molten agar (cooled to ~45-50 °C) into sterile Petri dishes.
2. Add sample (diluted bacterial suspension) by spread/dip; e.g., 1 mL per plate when using pour-plate counts.
3. Rotate plate to distribute inoculum; let solidify 5-10 min on a flat surface.
4. Incubate: typically 35 °C for ~24 h in 5% CO2 (or 30 °C up to 72 h) depending on strain.

#### For Broth

1. Inoculate MRS Broth with sample.
2. Incubate 35 °C for ~3 days, or 30 °C for ~5 days, under aerobic conditions (CO2 enrichment optional per strain).
3. Subculture to solid medium as needed for isolation and enumeration.

'},
{'slug':'gel','title':'Gel Electrophoresis','text':'

### Gel Electrophoresis

##### Equipment

* Casting tray
* Well combs
* Voltage source
* Gel box
* UV light source
* Microwave

##### Reagents

* 10x TAE (ThermoFisher)
* [Agarose](https://www.thermofisher.com/order/catalog/product/R0491)
* GelGreen Nucleic Acid Gel Stain

##### Protocol

### Preparing TAE

Dilute 10x TAE Buffer with water for 1x TAE concentration.

### Pouring a Standard X% Agarose Gel

1. Measure X g of agarose.
2. Mix agarose powder with 100 mL 1x TAE in a microwavable flask.
3. Microwave for 1-3 min until the agarose is completely dissolved (but do not overboil the solution, as some of the buffer will evaporate and thus alter the final percentage of agarose in the gel. Many people prefer to microwave in pulses, swirling the flask occasionally as the solution heats up).
4. While dissolved, add X mL of GelGreen Nucleic Acid Gel Stain (add to molten agarose at 1x final concentration).
5. Pour the agarose into a gel tray with the well comb in place, ensuring no bubbles.
6. Place newly poured gel at 4 °C for 10-15 mins OR let sit at room temperature for 20-30 mins, until it has completely solidified.

### Conducting Gel Electrophoresis

1. Add a loading buffer to each of your DNA samples and mix well. Use 5 µL of loading buffer per 25 µL of sample.
2. Once solidified, place the agarose gel into the gel box (electrophoresis unit).
3. Fill the gel box with 1x TAE (or TBE) until the gel is covered.
4. Carefully load your samples into the additional wells of the gel.
5. Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage.
6. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
7. Using any device that has UV light, visualize your DNA fragments. The fragments of DNA are usually referred to as bands due to their appearance on the gel.

'},
{'slug':'crude-lysate','title':'Cell Lysis','text':'

##### Protocol

### Buffer & Reagents (keep everything cold, 2–8 °C)

* **Lysis buffer (typical, cytosolic proteins):** 50 mM Tris-HCl pH 7.5–8.0, 150 mM NaCl, 5–10% glycerol
  Optional: 1 mM DTT (or 2 mM β-ME), 1 mM MgCl2, 0.1–1 mM EDTA\*
* **Protease inhibitor:** 1 tablet/10–50 mL (EDTA-free if you need metalloproteins).
* **DNase I:** 5–10 µg/mL (add with MgCl2) to reduce viscosity post-lysis.
* **Clarification supplies:** ice bucket, 0.45 µm prefilter (optional), centrifuge bottles.

*\*If you will purify a His-tagged metalloprotein, avoid EDTA.*

### Prep & Harvest

1. Grow culture to OD600 ~0.6–1.0 (or as required for expression). Chill culture on ice 10–15 min to slow proteolysis.
2. Pellet: 4,000–6,000 ×g, 10–15 min, 4 °C.
3. Resuspend pellet in cold lysis buffer at 3–5 mL per gram wet pellet. Add protease inhibitors. Keep on ice.

### Set Up Disruptor

* Clean/flush the system (water → 20–30% ethanol → water) if needed.
* Prime with a small volume of lysis buffer to remove air.
* Cooling: Use built-in heat exchanger, inline chiller, or coil tubing in an ice bath on the outlet. Aim to keep outlet <10 °C.

### Operating Parameters (typical for *E. coli*)

* **Pressure:** 10–20 kpsi (~70–140 MPa). Start at 10–12 kpsi; increase if lysis is poor.
* **Passes:** 1–3 passes. Most *E. coli* lyse >90% by 2 passes.
* **Flow rate:** As per instrument (e.g., 30–120 mL/min). Avoid cavitation; maintain steady back-pressure.
* **Viscosity control:** Add DNase I to the collection vessel at start of run (final 5–10 µg/mL + 1–5 mM MgCl2).

### Lysis Procedure

1. Prefilter the resuspension through 0.45 µm to catch clumps that can clog valves.
2. Load the cold cell slurry into the feed reservoir.
3. Run Pass 1 at 10–12 kpsi; collect into a pre-chilled bottle containing DNase I/MgCl2. Gently swirl to mix.
4. Assess quickly: take 50–100 µL for either:
   • Phase-contrast check (ghost cells vs intact), or
   • Quick OD drop/viscosity check with pipetting feel.
5. Run Pass 2 (and Pass 3 if needed) at 12–18 kpsi. Keep outlet cold. Avoid foaming.
6. Hold on ice 10 min to let DNase act and viscosity drop.

### Clarification

1. Centrifuge 15,000–20,000 ×g, 20–30 min, 4 °C to remove debris.
2. Save supernatant (crude lysate). For very fine debris, polish with 0.45 µm → 0.22 µm filtration if downstream requires.
3. Snap-sample for SDS-PAGE (pre/post lysis) to document efficiency.

'}
]
}
] %}

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![Results Banner](https://static.igem.wiki/teams/5659/header/results.webp)

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* [Overview](#overview-r)
* [Raw Data](#raw-data)
* [Data Analysis](#data-analysis)
* [Graphical Analysis](#graph-analysis)
* [Statistical Analysis](#stat-anaylsis)
* [Conclusion](#conclusion)
* [Future Directions](#future-direction)

## Overview

This page summarizes the key findings from our experimental analysis of IL-10 stability and outlines how these results guide our next steps. Our goal was to understand how environmental factors as well as engineered point mutations influence IL-10’s ability to remain stable and maintain its functional concentration.

Through statistical comparisons of experimental conditions, we identified significant differences that revealed where IL-10 is most prone to dissociation and how specific mutations can improve or reduce its stability. These insights not only validated our experimental approach but also highlighted areas where further testing and refinement are needed.

By consolidating these findings here, we aim to provide a clear picture of what we have learned so far and how it informs our strategy moving forward. The following sections present the major conclusions drawn from our data and propose realistic future directions to enhance reproducibility, optimize protein engineering, and strengthen the potential therapeutic application of IL-10.

## Raw Data

![ Figure 1](https://static.igem.wiki/teams/5659//results/result1.webp)

**Figure 1. Our raw data with labels pasted onto a google sheet & color coded for clarity.** Two trials are run (in triplicates) for this ELISA plate, with the standard curve on the leftmost column and blanks in all right columns.

Two hours within adding the Stop Solution, we obtained the values listed above through reading the ELISA plate at an absorbance setting of 450nm. We color-coded each of the samples, distinguishing them by: 1) temperatures of 37C or 39C; 2) IL10 variants of original, mutation 1, and mutation 2; 3) solvents of lysate or supernatant; 4) concentration readings done in two different trial rounds. The leftmost column displays our standard curve, with the concentrations obtained through a serial dilution.

## Data Analysis

First, we converted the absorbance values into concentration values to get more precise and universally comparable results. To do this, we used the AAT Bioquest Four Parameter Logistic (4PL) Curve Calculator to generate the standard curve. Optimally, the background absorbance may be subtracted from all data points, but the absorbance in all blank wells was 0 so no data was processed first.

In order to plot the standard curve, we put in the data points of the absorbance (or often referred to as optical density), and set up the calculator options to carry out background correction in order to subtract the values by the smallest response (0.042) since the value in the well with no Human IL-10 should’ve had 0 absorbance. We also normalized the data, which divided all values by the largest, in order to keep the values of absorbance between 0 and 1.

![Figure 2](https://static.igem.wiki/teams/5659//results/result2.webp)

**Figure 2. The standards used as values to calculate our curve are input into the software.** The settings & parameters are listed above which include display of error bars, background correction, and normalization of values.

![ Figure 3](https://static.igem.wiki/teams/5659//results/r3.webp)

**Figure 3. Our curve obtained through the standard absorbance data.** Regression results are also displayed to show how to calculate additional absorbance values into concentrations.

We then used this equation to calculate the concentration for each well. However, as can be seen in the equation, the y-intercept was 0.0878, and any absorbance value lower than that would not be able to give a concentration. For those wells (highlighted in purple), we set the concentration as 0.

![ Figure 4](https://static.igem.wiki/teams/5659//results/r4.webp)

**Figure 4. Restructured table of absorbance values.** The values in purple could not yield a concentration as they were below the y-intercept value of 0.0878.

![ Figure 5](https://static.igem.wiki/teams/5659//results/r5.webp)

**Figure 5. Table of calculated concentrations from the 4PL curve.**Notice that the wells originally highlighted in purple were set to 0.

## Data Analysis

We split the IL-10 variants into the two different trial rounds and a sum of both trial rounds. Using programming software R, we first graphed the concentration of the IL-10 variants into boxplots to visualize the data distribution and compare them. We also graphed a bar graph with error bars of 95% confidence intervals.

![ Figure 6](https://static.igem.wiki/teams/5659///results/r6.webp)

**Figure 6. Boxplot of concentrations of IL-10 variants.** On the x-axis are the types of IL-10 (temperature, lysate or supernatant, mutation number, and trial V or L). The trials with no labels of V or L are the average of the two. The y-axis marks the concentration.

![ Figure 7](https://static.igem.wiki/teams/5659//results/r7.webp)

**Figure 7. Bar graph of mean concentrations of IL-10 variants with error bars of 95% confidence interval.**

## Statistical Tests

With R, we conducted a one-way ANOVA to compare the mean concentrations of all of the IL-10 variants, and the p-value of **1.69E-5.** This is a very small p-value, signifying that we can **reject the null.**

![ Figure 8](https://static.igem.wiki/teams/5659//results/r8.webp)

**Figure 8. One way ANOVA outputs.** We can reject the null with a given p-value of 1.69E-5.

Since this only showed whether there is at least one pair that has a significant difference in its mean concentration, we conducted a Tukey’s Honestly Significant Difference (TukeyHSD) post-hoc test to see which pairs had the significant difference. However, none of the adjusted p values were <0.05, which we defined as statistically significant. We theorize that it likely has to do with our small sample size.

Thus, we conducted pairwise t-tests with an adjustment in p with the Benjamini-Hochberg (BH) procedure for all of the IL-10 variants. p<0.05 for t-tests between:

* 37L0 and 37S0
* 37L0 and 39S2
* 39L2 and 37S0
* 39L2 and 39S2
* 37S0 and 37S1
* 37S0 and 39S0
* 37S1 and 39S2
* 39S0 and 39S2

and more between the overall vs different trial rounds. For example, 37L0 and 37L2\_L or 37L0 and 37S0\_L. We did not deem these to have significant meaning so we did not count all of them. However, we have provided a comprehensive test between all different groups below as listed:

![ Test result 1](https://static.igem.wiki/teams/5659//results/test1.webp)
![ Test result 2](https://static.igem.wiki/teams/5659//results/test2.webp)

## Conclusion

From the eight pairwise comparisons that produced statistically significant differences (p < 0.05), three central conclusions emerge regarding the behavior of IL-10 under varying conditions. These findings highlight how both environmental factors and engineered mutations influence the protein’s stability and binding efficiency.

First, our data demonstrate that temperature plays a measurable role in IL-10 dissociation. When the original IL-10 obtained from the supernatant was incubated at either 37 °C or 39 °C, we observed a significant difference in the concentration of IL-10 bound to its antibody. This pattern suggests that even a modest increase in temperature can disrupt IL-10’s conformational stability, reducing its capacity to maintain interactions with the antibody. Given that IL-10’s therapeutic effectiveness depends on its stability and bioavailability, these results underscore the importance of temperature control during both experimental assays and potential clinical applications.

Second, the results reveal that the source of the IL-10—whether collected from the lysate or the supernatant—significantly influences its concentration. For the original IL-10 incubated at 37 °C, samples derived from lysate exhibited markedly different concentrations than those obtained from the supernatant. A similar pattern was observed for the IL-10 variant carrying mutation 2 when incubated at 39 °C. This difference likely reflects how the cellular localization of the protein—either retained within cells or secreted into the surrounding medium—affects its solubility, folding state, or exposure to degradation. These findings highlight that not only the genetic sequence but also the source of the protein must be considered when assessing IL-10 stability.

Finally, our analysis indicates that engineered mutations can substantially alter IL-10’s concentration compared to the original protein. In particular, IL-10 with mutation 1 displayed a significantly different concentration from the original protein when collected from the supernatant and incubated at 37 °C. Likewise, IL-10 with mutation 2 differed significantly from the original when collected from the supernatant and incubated at 39 °C. These observations suggest that structural modifications introduced by these mutations may enhance or diminish IL-10’s stability under specific thermal conditions, thereby impacting its recovery and functional activity.

Taken together, these findings highlight the complex interplay between environmental conditions, protein source, and engineered mutations in determining IL-10’s stability and binding properties. Understanding these relationships is crucial for optimizing experimental protocols and guiding future therapeutic development of IL-10 variants with improved resilience to physiological stressors.

## Future Directions

Building on what we learned this season, there are several realistic steps we could take to further improve the stability, reproducibility, and delivery of IL-10. These directions would allow us to strengthen both the scientific foundation of our work and its potential therapeutic application.

- 1. Expanding Protein Engineering

We could explore additional point mutations guided by computational tools such as Rosetta or ThermoMPNN to identify substitutions that improve IL-10’s thermal stability and resistance to degradation. We could also focus on the dimer interface—where IL-10 is most prone to dissociation—by introducing targeted mutations or engineered disulfide bridges. These incremental modifications could lead to more robust variants that are better suited to real-world conditions.

- 2. Deepening Biophysical Characterization

To better understand why certain variants behave differently, we could perform additional structural and biophysical analyses. Techniques such as circular dichroism, differential scanning calorimetry, and size-exclusion chromatography could help us monitor folding and aggregation. We could also use surface plasmon resonance (SPR) to measure how mutations affect IL-10’s binding to antibodies or receptors at different temperatures.

- 3. Enhancing Experimental Reproducibility

We could conduct future tests with more biological and technical replicates to reduce variability and strengthen statistical significance. Standardizing assay conditions—such as incubation times, sample handling, and ELISA workflows—would help reduce experimental noise. Regular calibration of instruments and the use of consistent internal controls could further improve reproducibility and confidence in the data.

- 4. Optimizing Production and Secretion

Since we ultimately envision probiotic delivery, we could explore expressing and secreting IL-10 variants in Lactobacillus plantarum—our intended chassis—instead of focusing only on E. coli. Testing different signal peptides and secretion systems could also help us improve protein yield and stability in the supernatant, which was an important variable in our current experiments.

- 5. Testing Therapeutic Functionality

To move closer to demonstrating therapeutic potential, we could evaluate the IL-10 variants in cell-based assays that measure suppression of pro-inflammatory cytokines, such as TNF-α and IL-6. As a longer-term goal, we could explore testing these variants in gut organoid models to better understand their performance in a more physiologically relevant environment.

- 6. Advancing Delivery Strategies

We could also integrate our protein engineering with more sophisticated delivery approaches. For example, coupling IL-10 variants with inflammation-responsive promoters in probiotics could ensure that the protein is secreted only during flare-ups. We could further examine protective encapsulation methods—such as alginate or other biocompatible polymers—to maintain protein stability during storage and transport.

In summary, these future directions focus on building a stronger foundation for IL-10 engineering while making our experiments more reproducible and clinically relevant. By combining computationally guided improvements, standardized testing, optimized production in probiotics, and innovative delivery strategies, we could make meaningful progress toward a practical, stable IL-10–based therapeutic for inflammatory bowel disease.

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