iGEM 2025 — Team hk-hcy-pcms

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# NOTEBOOK

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# ENGINEERING

### Engineering

* [Abstract](#section1)
* [Cycle 1](#section2)
* [Cycle 2](#section3)
* [Conclusion](#section4)
* [Reference](#section5)

## Abstract

This project aims to develop a multi-target eyedrop that can tackle glaucoma. We have iteratively
engineered our strategies over the past months to develop two fusion proteins, FT and BC, aimed at
addressing the two primary causes of open-angle glaucoma: high intraocular pressure and retinal ganglion
cell apoptosis. By using the engineering cycles, we will describe our designing, building, testing, and
learning phases. In the next section, we will explain how we applied the principles of engineering
design thinking to successfully finish our project.

## Cycle 1

### Design 1.1

In this first cycle, our primary objective was to establish a foundational design for a fusion protein utilizing PEP-1 and FWT, which consists of FRATtide, Wnt3α,
and TP1. Additionally, we incorporated another fusion peptide known as BC, which consists of BDNF and CDR1, designed to help prevent apoptosis and increase retinal ganglion cells’
survival rates. We constructed a plasmid for the respective fusion peptides, focusing on how each element contributes to the overall functionality of the fusion protein.

We also decided to incorporate the recombinant plasmid pUC18 into our strategy primarily for gene cloning and amplification. pUC18 is well-suited for this purpose due to its small size and high copy number, which enables efficient replication in E. coli DH5-α cells. [1]

For gene and protein expression, we utilized the DH5-α and BL21 strains. DH5-α was selected for its high transformation efficiency and stable plasmid maintenance [2], making it ideal for cloning and amplifying plasmid DNA. This strain is particularly effective for introducing recombinant DNA constructs, including fusion peptides, into cells for plasmid amplification. Moreover, BL21 was chosen for its capability of high-level protein expression, especially in the production of recombinant proteins. The T7 RNA polymerase gene in BL21 facilitates strong transcription of T7 promoter-driven target genes, allowing us to effectively express our fusion peptide constructs and obtain the desired peptides. [3]

![Team Photo](https://static.igem.wiki/teams/5641/master/wetlab/fwt-puc.webp)

Fig 1 FWT Benchling plasmid design (pUC18).

![Team Photo](https://static.igem.wiki/teams/5641/master/wetlab/bc-puc.webp)

Fig 2 BC Benchling plasmid design (pUC18).

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/fwt-plasmid-flow.webp)

Fig 3 FWT flow

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/bc-plasmid-flow.webp)

Fig 4 BC flow

The design process involved careful consideration of the roles of each component. In FWT, FRATtide plays a crucial role in inhibiting GSK3, thereby contributing to the higher expression of Axin2 and MMPs. Both of them are responsible for degrading the ECM resistance of aqueous humor. Wnt3α is one of the members of the Wnt signal family, which can benefit from the Wnt/β-catenin signaling pathway functions. TP1, a peptide extracted from ginsentide, was included for its known properties in enhancing NO production and muscle relaxation. In BC, BDNF activates PI3K, MAPK, and PLCy pathways, which can prevent cell apoptosis; and also lower the oxidative stress and eventually facilitate neuroprotection. On the other hand, CDR1 can inhibit caspase-3 indirectly to minimize the RGC death rate.

### Build 1.2

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/wnt3a.webp)

Fig 5 Alphafold design for Wnt3α.

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/pegofwnt3a.webp)

Fig 6 Alphafold position error graph of Wnt3α.

Following the design phase, we moved into the lab implementation. We constructed the gene and transformed it into DH5-α and E. coli for their respective functions.
Both the transformation of BC and FWT succeeded.

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/okok/screenshot-2025-10-08-at-17-11-12.avif)

Fig 7 Culture of transformed DH5-α.

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/okok/screenshot-2025-10-08-at-17-11-00.avif)

Fig 8 Culture of transformed BL21.

We will then perform plasmid purification and protein purification to obtain high-quality, purified plasmid and protein. Initial results from both gel electrophoresis and SDS-PAGE
suggested that our fusion protein was expressed successfully, leading us to believe that our design was functioning as intended.

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/fwtgel.webp)

Fig 9 FWT Gel electrophoresis.

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/bcgel-1.webp)

Fig 10 BC Gel electrophoresis.

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/okok/sds-new.avif)

Fig 11 SDS-PAGE result of FWT and BC.

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/proteinpurification-1.webp)

Fig 12 Protein purification.

### TESTING 1.3

Subsequent testing revealed significant limitations in the performance of the evaluated components. An ELISA assay was performed to assess binding affinity. Results from the ELISA are generally interpreted based on two key criteria: visual color intensity and the quality of the standard curve. Ideally, the intensity of yellow color developed in the assay should be directly proportional to the amount of target protein in the sample, indicating a successful enzymatic reaction. Furthermore, the standard curve must exhibit a high degree of fit to the data points, which is quantified by the coefficient of determination (R²). An R² value of 0.99 is generally expected for reliable quantification; this shows that the assay measures target analyte concentrations reliably over the tested range. A lower R² value would suggest subpar assay performance, necessitating troubleshooting and a follow-up round of testing.

In our initial ELISA testing, an ideal standard curve quality (R²) was obtained, confirming the efficacy of its design and functionality. The presence of robust protein for the BC component was demonstrated, as evidenced by a strong yellow signal, providing a solid foundation for further development of this component. In contrast, the FWT component showed no detectable protein presence, indicated by the absence of a yellow signal and lack of absorption.

### LEARN 1

As a response to these findings, we decided to modify our approach. We hypothesized that the presence of Wnt3α was detrimental to the protein's ability to fold correctly and bind to its intended receptors.

Additionally, we hypothesized the need for a more effective E. Coli strain for more protein expression. Consequently, we removed Wnt3α from our construct and replaced it with PEP-1 FT,
and we replaced BL21 with SHuffle cell, which we anticipated would enhance both the folding and binding potential of the fusion protein cycles, and increase the amount of proteins expressed,
respectively.

Finally, the recombinant plasmid pUC18 proved to have a better cloning efficiency, but the protein yields from the results were insufficient for subsequent experiments. This signals a need for a different recombinant plasmid, designed to increase protein expression.

## Cycle 2

### Design 2.1

In the second cycle, we started by redesigning our fusion peptide FWT by removing Wnt3α, leading to a modified fusion peptide now referred to as FT. We conducted experiments with BC without further modifications, as it yielded positive results in all tests during cycle 1. Additionally, while BL21 could express our protein, the yield was insufficient. Therefore, we replaced BL21 with the more effective SHuffle cell for improved expression.

Additionally, we designed a new recombinant plasmid, pET-IDT, dedicated to enhancing protein expression levels. It excelled in expression due to its strong IPTG-inducible T7 promoter and lac operator system. This vector, when paired with specialized host strains like E. coli SHuffle cells, supported enhanced protein folding and post-translational modifications crucial for our eyedrop. [4]

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/screenshot-2025-10-08-at-16-56-50.avif)

Fig 13 FT Benchling plasmid design (pet-IDT).

![Team Photo](http://static.igem.wiki/teams/5641/master/engineering/screenshot-2025-10-08-at-16-57-06.avif)

Fig 14 BC Benchling plasmid design (pet-IDT).

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/ft-plasmid-flow.webp)

Fig 15 FT flow

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/bc-plasmid-flow.webp)

Fig 16 BC flow

### Build 2.2

We designed SHuffle cells to avoid periplasmic export or refolding from inclusion bodies by directly generating active, disulfide-bonded proteins (such as antibodies, enzymes, or cytokines) in the cytoplasm. Compared to common strains like BL21, SHuffle cells offer improved folding accuracy for proteins with multiple disulfide links, leading to enhanced activity and solubility. In SHuffle cells, they express disulfide bond isomerase DsbC, which provides an oxidizing cytoplasmic environment that facilitates the production and repair of disulfide bonds within the cytoplasm, unlike BL21.

The synthesis of properly folded and functional proteins—including those that are frequently inactive or insoluble when expressed in BL21—is made possible by this environment. Furthermore, SHuffle provides multiple alternatives for gene expression by supporting T7 promoters. For disulfide-bonded proteins, the yields from SHuffle cells are frequently on par with or superior to those from conventional periplasmic expression systems. This makes SHuffle particularly advantageous for expressing complex fusion peptides, such as those in your glaucoma eyedrop project, as it ensures the production of these peptides with correct structural conformation, increased solubility, and higher therapeutic activity. [5]

To enhance the delivery and therapeutic efficacy of our fusion peptide drugs within the eye, we utilized nanocarrier liposomes as advanced delivery vehicles. These liposomes facilitate improved penetration through ocular barriers, ensuring that the eyedrop reaches deeper target tissues with higher efficiency. This targeted delivery system also helps reduce systemic exposure and off-target effects, thereby minimizing potential side effects and increasing the safety profile of the treatments. Specifically, for the FT drug, we engineered the liposomes to carry and direct the peptides toward pathways involving CTGF and LRP5/6, which play key roles in ocular health and disease modulation. For the BC drug, liposomes target the NGF-TrkA signaling axis, enhancing selective receptor binding and downstream therapeutic effects. Upon reaching the target cells, these nanocarrier liposomes interact with specific receptors on the cell surface. This interaction triggers receptor-mediated endocytosis, a process in which the cell membrane engulfs the liposome-drug complex, internalizing it into the cell. This mechanism ensures efficient intracellular delivery of the drug, allowing it to reach its site of action within the cell. By leveraging these tailored liposomal delivery systems and receptor-mediated uptake, we achieve more precise drug localization, sustained release, and improved bioavailability, all of which contribute to maximizing clinical benefits while maintaining safety in treating eye conditions. [6][7][8]

Specifically, for the FT drug, we engineered liposomes to carry and direct the peptides toward pathways involving CTGF and LRP5/6, which play key roles in ocular health and disease modulation.

Finally, for the BC drug, liposomes target the NGF-TrkA signaling axis, enhancing selective receptor binding and downstream therapeutic effects. By leveraging these tailored liposomal delivery systems, we achieve more precise drug localization, sustained release, and improved bioavailability, all of which contribute to maximizing the clinical benefits while maintaining safety in the treatment of eye conditions.

We will conduct the Cycle 2 experiments in the same manner as Cycle 1, employing SDS, gel electrophoresis, and ELISA to rigorously assess our protein. Once we've addressed all potential improvements in our experiments, we'll proceed to cell line engineering.

We conducted gel electrophoresis, and the results indicate that our DNA expression in bacterial cells for the newly designed plasmid was successful.

![Team Photo](https://static.igem.wiki/teams/5641/master/edu/ww/screenshot-2025-10-07-164952.webp)

Fig 17 Culture of transformed DH5-α.

![Team Photo](https://static.igem.wiki/teams/5641/master/edu/ww/screenshot-2025-10-07-171455.webp)

Fig 18 Culture of SHuffle cells.

As round 1, the E. coli containing our recombinant plasmids were cultured, lysed and underwent protein purification.

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/gel-2.webp)

Fig 19 BC and FT Gel electrophoresis.

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/sds-2.webp)

Fig 20 SDS-PAGE result of FT and BC.

Repeating the experiments from the initial cycle, we examined the presence of our recombinant plasmid using SDS-PAGE and gel electrophoresis. The results from SDS-PAGE confirmed the presence of our protein once again, and both transformations for BC and FT were successful. This success allows us to move forward with additional testing, which includes conducting ELISA kits. Furthermore, we observed a higher level of protein expression in the culture of SHuffle cells compared to that of BL21, which supports our hypothesis.

### Testing 2.3

In the BCA test, we will test the protein concentration in our drugs.

Two BCA assays were performed: one before protein refolding and one after protein refolding. Both sets of results were within the expected range. From Figure 21, it can be concluded that the proteins possessed a high concentration prior to refolding. Figure 22 indicates the optimal pH condition under which the proteins refolded most effectively, yielding the highest concentration. The purple coloration observed in the wells corresponds to protein concentration, as the intensity of this purple color, measured by absorbance at 562 nm, is directly proportional to the amount of protein present in the sample. The observed gradual change in color intensity across the dilution series confirms that the results meet the expected trend.

![Team Photo](https://static.igem.wiki/teams/5641/master/wetlab/bca1.webp)

Fig 21 BCA RESULTS BEFORE PROTEIN REFOLDING

![Team Photo](https://static.igem.wiki/teams/5641/master/wetlab/bca2.webp)

Fig 22 BCA RESULTs AFTER PROTEIN REFOLDING

In ELISA tests, we will again test the binding affinity of the fusion peptides.

The results revealed strong protein presence in BC as seen visually by the yellow present in the result pictures; on top of that, the standard curve quality is optimal, for PI3K, it has a R² value of 0.997, for ANP32A, it has a R² value of 0.998, for TrkB, it has a R² value of 0.9967, further validating the usage of BC.

The results for FT followed suit, first with intense yellow colors in its results, paired with the desirable standard curve quality as well, for PI3K, it has a R² value of 0.997, for NOS, it has a R² value of 0.9991, for GSK3, it has a R² value of 0.9972, which means FT had successful results for all three experiments.

We have conducted absorbance tests using the ELISA kit assays, and the results are presented below.

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/elisa-anp32a.webp)

Fig 23 ELISA results of ANP32A

![Team Photo](https://static.igem.wiki/teams/5641/master/edu/ww/dday/anp32a.avif)

Fig 24 ELISA graph of ANP32A

![Team Photo](https://static.igem.wiki/teams/5641/master/edu/ww/dday/image-1.avif)

Fig 25 ELISA results of TrkB, PI3k, NOS, GSK3.

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/elisatrkb2.avif)

Fig 26 ELISA graph of TrkB.

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/elisapi3k.webp)

Fig 27 ELISA graph of PI3K.

![Team Photo](https://static.igem.wiki/teams/5641/master/edu/ww/dday/nos.avif)

Fig 28 ELISA graph of NOS.

![Team Photo](https://static.igem.wiki/teams/5641/master/edu/ww/dday/gsk3.avif)

Fig 29 ELISA graph of GSK3.

After confirming the binding affinity of our peptide via ELISA, the next step is to perform functional testing on actual cell lines. We plan to evaluate the therapeutic effects of the peptide on mouse retinal ganglion cells and human trabecular meshwork cells (HTMC). This will determine whether the peptide exerts its therapeutic potential in relevant human cellular models.
We will apply our fusion peptide BC to mouse retinal ganglion cells (RGCs) and assess apoptosis rates using the Annexin V-FITC/PI Apoptosis Detection Kit. Additionally, FT will be administered to human trabecular meshwork cells (HTMCs) to evaluate nitric oxide (NO) production, measured by the fluorescent probe DAF-FM DA.

### Learn 2

The successful results from several different tests confirm our hypothesis that our previous fusion peptide, FWT, was negatively affected by Wnt3α.

In addition to the successful results for our fusion peptides, the fact that the optimized culture yields a significantly higher amount of protein than the BL21 culture we performed in cycle 1 shows how the structure of SHuffle cells affects the rate of expression and, consequently, the protein's ultimate yield.

Additionally, in Cycle 1, pET-IDT serves as an ideal replacement for pUC18 because it enables the reliable synthesis of therapeutic proteins that are both biologically active and correctly folded. Several of our peptides require the production of disulfide bonds for action, and the oxidative cytoplasmic environment provided by E. coli SHuffle strains improves this process.

Through these two rounds of experiments, we have discovered that plasmids offer distinct advantages based on the vector and host system employed. Plasmids are natural extrachromosomal DNA elements found in bacterial cells, providing genetic advantages such as antibiotic resistance. In our project, we initially used the pUC18 plasmid in conjunction with the E. coli strain DH5-α for cloning, while utilizing BL21 cells for protein expression. In Cycle 2, we replaced pUC18 with the pET-IDT plasmid for protein expression.

#### pUC18

Small plasmid size (2.7 kb) allows easy manipulation and cloning of large DNA fragments.

High copy number in E. coli DH5-α enables robust plasmid replication and gene amplification.

#### pET-IDT

Strong IPTG-inducible T7 promoter enables tightly regulated, high-level expression of therapeutic proteins in specialized strains like E. coli SHuffle.

Lac operator allows precise transcriptional control, minimizing unwanted basal expression of potentially toxic proteins.

Compatible with host strains promoting disulfide bond formation and proper protein folding, critical for biologically active glaucoma therapeutics.

Ultimately, these results further demonstrate that our approach of constructing fusion peptides as a solution for glaucoma is both feasible and achievable.

## Conclusion

Throughout the two cycles of our engineering project, we have gained valuable insights into the challenges we faced and the complexities of peptide modification.

After modifying the fusion peptide by removing one of the components from our initial fusion protein’s design, Wnt3α, and replacing BL21 with SHuffle cells for protein synthesis, we successfully increased the stability of our peptide and the amount of proteins expressed. The function of our peptide has also been modified. The successful lab results, including the ELISA results, indicate the improvement of our peptide design.

In cycle 2, we switched to pET-IDT, upon realizing the deficiencies of pUC18, which offers strong, IPTG-inducible T7 promoter-driven expression and works well in DH5-α for cloning and Shuffle cells for high-yield protein production. This system ensures robust, tightly controlled expression and proper protein folding, optimizing production of active therapeutic proteins for our glaucoma treatment.

These modifications deepened our insight into peptide dynamics and highlighted the importance of flexibility in experimental design. Throughout the project, we faced challenges that sharpened our critical thinking and problem-solving abilities, teaching us to overcome obstacles with innovative solutions. This ongoing process not only advanced our technical skills but also reinforced the vital roles of resilience and teamwork in scientific research. Together, these experiences strengthened both our scientific understanding and collaborative approach, laying a strong foundation for future work.

## Reference

[1] Xavier MA, Kipnis A, Torres FA, Astofi-Filho S. New vectors derives from pUC 18 for clonig and thermal-induced expression in Escherichia coli. Braz J Microbiol. 2009 Oct;40(4):778-81. doi: 10.1590/S1517-83822009000400007. Epub 2009 Dec 1. PMID: 24031424; PMCID: PMC3768577.

[2] Al-Janabi SS, Shawky H, El-Waseif AA, Farrag AA, Abdelghany TM, El-Ghwas DE. Stable, efficient, and cost-effective system for the biosynthesis of recombinant bacterial cellulose in Escherichia coli DH5-α platform. J Genet Eng Biotechnol. 2022 Jun 23;20(1):90. doi: 10.1186/s43141-022-00384-7. PMID: 35737166; PMCID: PMC9226222.

[3] Heyde, S.A.H., Nørholm, M.H.H. Tailoring the evolution of BL21(DE3) uncovers a key role for RNA stability in gene expression toxicity. Commun Biol 4, 963 (2021).

[4] Dong L, Zhang X, Yu C, Ren J, Hou L, Fu L, Yi S, Chen W. Expression and purification of recombinant proteins based on human prostate stem cell antigen and heat shock protein-70. Exp Ther Med. 2013 Apr;5(4):1161-1164. doi: 10.3892/etm.2013.967. Epub 2013 Feb 20. PMID: 23596484; PMCID: PMC3627452.

[5] Lobstein J, Emrich CA, Jeans C, Faulkner M, Riggs P, Berkmen M. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microb Cell Fact. 2012 May 8;11:56. doi: 10.1186/1475-2859-11-56. Erratum in: Microb Cell Fact. 2016 Jul 13;15(1):124. doi: 10.1186/s12934-016-0512-9. PMID: 22569138; PMCID: PMC3526497.

[6] Mehta HM, Woo SB, Neet KE. Comparison of nerve growth factor receptor binding models using heterodimeric muteins. J Neurosci Res. 2012 Dec;90(12):2259-71. doi: 10.1002/jnr.23116. Epub 2012 Aug 18. PMID: 22903500; PMCID: PMC3674885.

[7] Marlin MC, Li G. Biogenesis and function of the NGF/TrkA signaling endosome. Int Rev Cell Mol Biol. 2015;314:239-57. doi: 10.1016/bs.ircmb.2014.10.002. Epub 2014 Nov 18. PMID: 25619719; PMCID: PMC4307610.

[8] Mercurio, S., Latinkic, B., Itasaki, N., Krumlauf, R., & Smith, J. C. (2004). Connective-tissue growth factor modulates WNT signalling and interacts with the WNT receptor complex.

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![IMG](https://static.igem.wiki/teams/5641/master/homepage/wet-lab.webp)

# EXPERIMENTS

Overview

Protocol

Materials

### Experiments

* [Experiment Overview](#section1)
* [Protocol](#section2)
* [Material and Method](#section3)
* [Safety](#section4)

## Experiment Overview

After designing our gene construct, we embarked on our laboratory work to validate and optimize our product. We developed a series of experiments to assess different aspects of our project.

First, we performed polymerase chain reaction (PCR) to amplify our target DNA sequence, followed by agarose gel electrophoresis to verify the correct gene length. The plasmid of our validated targeted gene included will be transformed into DH5α E. coli cells for plasmid duplication. The duplicated plasmids were then extracted to validate their length through agarose gel electrophoresis also. Then, the plasmids were transformed into BL21 E. coli cells for protein expression. Subsequently, we purified the expressed protein and conducted SDS-PAGE analysis to evaluate protein size and purity.

To determine the optimal dosage of our protein product, we utilized ELISA assays. In the final phase of our experimental workflow, we collaborated with CUHK to carry out cell line experiments. These studies aimed to investigate the biological effects of our drug in a cellular context.

Throughout the process, we systematically tested various experimental conditions and formulations to optimize performance. Through iterative adjustments and careful analysis, we strived to achieve the most effective and reliable results.

## Protocol

PCR

Polymerase Chain Reaction (PCR) is an effective laboratory technique for DNA amplification. It used to rapidly create millions to billions of copies of a specific segment of DNA.

1. 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 entire reaction should be mixed again to ensure homogeneity. Collect all liquid to the bottom of the tube with a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.
2. Add different components into the sterile thin-walled PCR tube according to the proportion below:
   1. First Round PCR

      |  |  |
      | --- | --- |
      | 10 μM Forward Primer | 1μl |
      | 10 μM Reverse Primer | 1μl |
      | DNA | 1μl |
      | One *Taq* 2X Master  Mix with Standard Buffer | 25μl |
      | Nuclease-free water | 22μl |
   2. Second Round PCR

      |  |  |
      | --- | --- |
      | UTR mix | 3μl |
      | First Round PCR Product | 5μl |
      | One *Taq* 2X Master  Mix with Standard Buffer | 25μl |
      | Nuclease-free water | 17μl |
3. Put the tubes into the PCR machine and start it.

   Thermocycling condition for a routine PCR

   | STEP | TEMP | TIME |
   | --- | --- | --- |
   | Initial Denaturation | 95°C | 30 seconds |
   | 25-35 Cycles | 95°C  45-68°C\*  68°C | 15-30 seconds  15-60 seconds  1 minute/kb |
   | Final Extension | 68°C | 5 minutes |
   | Hold | 4-10°C |  |

▼

Transformation

Transformation is the process of introducing foreign DNA — such as a plasmid into a host cell. We used BL21, a type of e-coli. to take up the plasmid and express new genetic material.

1. Thaw competent cells on ice.
2. Chill approximately 5 ng (2 μl) of the ligation mixture in a 1.5 ml microcentrifuge tube.
3. Add 50 μl of competent cells to the DNA. Mix gently by pipetting up and down or flicking the tube 4–5 times to mix the cells and DNA. Do not vortex.
4. Place the mixture on ice for 30 minutes. Do not mix.
5. Heat shock at 42°C for 30 seconds. Do not mix.
6. Add 950 μl of room temperature media to the tube.
7. Place tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm selection plates to 37°C.
9. Spread 50–100 μl of the cells and ligation mixture onto the plates.
10. Incubate overnight at 37°C.

▼

Gel electrophoresis

Gel electrophoresis is a commonly used laboratory technique that separates charged molecules—such as DNA, RNA, or proteins—based on their size. In this method, an electric field drives the molecules through a porous gel matrix. Smaller molecules migrate faster through the gel's pores, while larger ones move slower, resulting in distinct bands that can be visualized for analysis.

1. Preparation of agarose

   1. Measure 0.3 g agarose powder
   2. Mix agarose power with 30mL 1X TAE buffer with a microwavable flask
   3. Microwave solution for 1 minute. Microwave for longer if there is undissolved agarose powder
   4. Let the agarose solution cool down to about 50°C, about 5 minutes.
   5. Add 3µl GelGreen® Nucleic Acid Gel Stain into the gel solution, and mix them well.
2. Pour the solution into the gel tray. Insert the comb into the top of the gel.
3. Wait for around 15 minutes to let the gel solidify.
4. Running gel electrophoresis

   1. Once the gel has solidified, carefully remove the comb by pulling it straight up.
   2. Place the agarose gel into the gel box.
   3. Fill the gel box with 1 X TAE buffer until the gel is covered.
   4. Prepare the sample by adding a loading buffer to each, with the ratio of 1:5.
   5. Load 15μL of DNA ladder into the first lane of the gel.
   6. Load samples into wells. Avoid bubbles.
   7. 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.
   8. Turn off the power and carefully remove the gel from the gel box.
   9. Image it with UV.

▼

Protein purification

Protein purification is a laborious process used to isolate a specific and targeted protein from a complex mixture.

1. Reagent preparation
   1. DTT Solution: Dissolve one vial of DTT (Solid) in 1 mL of ultrapure water. DTT solution must be prepared fresh before used
   2. Lysis Buffer: Mix Native Lysis Buffer with freshly prepared DTT solution at a 200:1 ratio.
   3. Wash Buffer: Mix Native Wash Buffer with freshly prepared DTT solution at a 200:1 ratio.
   4. Elution Buffer: Mix Elution Wash Buffer with freshly prepared DTT solution at a 200:1 ratio.
2. Add 20μL of Lysis Buffer into the bacteria with our DNA, and resuspend the bacteria in the lysate sufficiently, allowing for a slight vortex (to minimize bubbles).
3. Centrifuge at 4°C for 10 min.
4. Add 500 μL of 50% His tag Purification Resin (Reductant and Chelator Resin) mixed evenly, and slowly shake on a shaker at 4°C for 30 minutes to fully bind the target protein with His tag.
5. Centrifuge 10 s at 4°C to precipitate the gel, take 20 μL of the supernatant for subsequent detection, and discard the rest of the supernatant.
6. Add 100 μL Wash Buffer resuspension gel, centrifuge at 4°C 10 s, retention sample for subsequent detection, and discard the rest. Take 20 μL supernatant
7. Repeat step (6) with one more wash.
8. Add 20 μL of Elution Buffer and gently resuspend the gel. Centrifuge at 4°C 10 s to collect the supernatant and gel. The supernatant is the purified His-tagged protein of interest.
9. Repeat step (8) twice. Approximately 60 μL of purified protein samples were collected by co-elution.

▼

SDS-PAGE

SDS-PAGE is used to separate proteins based on their molecular weight. The proteins are loaded onto a polyacrylamide gel. When an electric field is applied, proteins migrate through the gel toward the positive electrode. We can identify the relative molecular weights by comparing the migration distance of proteins.

1. Place the gel in the SDS-PAGE stand
2. Place the stand with the gel in the SDS-PAGE apparatus bath
3. Fill the space between the gels with running buffer
4. Take off the combs of the gel (take it out gently)
5. Load the sample to each well
6. Do not use the wells at the left and right end of the gel
7. Fill the SDS-PAGE apparatus bath with running buffer to the bottom of the gel
8. Place the cover of the SDS-PAGE
9. Plug the cables to the SDS-PAGE apparatus power source
10. Turn off the SDS-PAGE power source
11. Set to constant voltage
12. Use 200 V
13. Press the Run button to start the electrophoresis
14. The voltage increases to 200 V from 0 V
15. For 4% staking gel and 12.5% separating gel the current reaches 50-60 mA and the power 10-12 W
16. The current then the power decreases in time but not reach zero (if it reaches zero see the troubleshoot)
17. For the state type of gel it takes 35 - 40 mins

▼

Cell culture

1. Thaw competent cells on ice
2. Add 1-5μl (10pg-100 ng) of plasmid (do not exceed 5μl for a 50μl cell aliquot)
3. Incubate on ice for 30 minutes
4. Heat-shock by placing in 42°C water bath for exactly 30 seconds
5. Place cells on ice for 2 minutes
6. Add 1mL pre-warmed LB or SOC medium
7. Shake incubate 37°C, 200rpm, 1 hour for outgrowth

▼

Cell lysis

If monitoring cell density by OD600, record final OD readings prior to harvesting.

1. Harvest cells by centrifugation at 16,000 × g for 10 minutes. For larger volumes, centrifuge for ≥30 minutes especially if at ≤10,000 × g. Discard the medium and, if necessary, weigh the wet cell pellet.
2. Store the pellet at -20°C or -80°C or process immediately.
3. Resuspend the cell pellet in NEBExpress E. coli Lysis Reagent by pipetting or vortexing briefly until the suspension is homogenous:
4. Use 0.025 - 0.075 mL of NEBExpress E. coli Lysis Reagent for every 1 UOD600 harvested. To calculate the UOD600, multiply the volume harvested by the OD600 reading. For example, a 5 mL culture harvested at OD600 1 gives 5 mL x 1.0 = 5 UOD600. In this example, 0.125 – 0.375 mL Lysis Reagent is required to lyse efficiently.
5. If harvested cells are weighed, use 5 mL of NEBExpress E. coli Lysis Reagent per 1 gram of cells.
6. Incubate the resuspended cells at room temperature for 10 - 20 min with gentle shaking, gentle rotation, or swirling. Lysis is usually visible with a clearance of the suspension.
7. Centrifuge the lysate at 16,000 x g for 10 min at 4°C to pellet the insoluble material and cell debris (30 min or longer for large volumes and lower speed).
8. Carefully transfer the supernatant into a sterile container for analysis or purification. This soluble fraction can be stored at 4°C for a few hours or -20°C or -80°C for longer term storage.
9. If needed, resuspend the insoluble pellet in 50 mM

▼

ELISA

ELISA is a widely used laboratory method for detecting and quantifying specific substances—typically proteins, antigens, or antibodies—in various biological samples such as blood, serum, or cell culture supernatants.

#### Reagent Preparation

1. Dilute the wash solution with deionized water (1:20)
2. Set standard wells and test sample wells. Add 50 μl standard to standard well.
3. Add 10 μl testing sample then add 40 μl sample diluent to testing sample well; blank well does not add anything.
4. Add 100 μl of HRP conjugate reagent to each well, cover with an adhesive strip and incubate for 60 minutes at 37°C.
5. Aspirate each well and wash, repeating the process four times total of five washes, wash by filling each well with Wash solution (400 μl) using a squirt bottle, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting. Invert the plate and blot it against clean paper towels.
6. Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light.
7. Add 50μl Stop solution to each well. The color in the wells should change from blue to yellow if the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8. Read the Optical Density (O.D.) at 450nm using a microtiter plate reader within 15 minutes.

#### Calculation of Results

1. This standard curve is used to determine the amount in an unknown sample. The standard curve generated by plotting the average O.D. (450 nm) obtained for each of the six standard concentrations on the vertical (Y) axis versus the corresponding concentration on the horizontal (X) axis.
2. First, calculate the mean O.D value for each standard and sample. All O.D values, are subtracted by the mean value of the zero standard before result interpretation. Construct the standard curve using graph paper or statistical software.
3. To determine the amount each sample, first locate the O.D. value on the Y-axis and extend a horizontal line to the standard curve, t the point of intersection, draw a vertical line to the X-axis and read the corresponding concentration
4. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result. Each user should obtain their own standard curve.
5. The sensitivity by this assay is 1.0 pg/ml

▼

Cell line

#### Cell Revival

1. Preheat water bath to 37°C.
2. Prepare one 15 ml centrifuge tube, add 5 ml complete medium containing 10% FBS, and preheat in 37°C water bath.
3. Wear protective goggles and gloves, take out the frozen cells from liquid nitrogen quickly and place them in the preheated water bath to increase thawing speed.
4. Transfer thawed cells to the prepared centrifuge tube, mix well, and centrifuge at 1000 rpm for 5 minutes.
5. Prepare a T-25 flask, write cell name and date, add 4 ml complete medium.
6. After centrifugation, discard supernatant, resuspend the cell pellet in 1 ml complete medium, transfer to T-25 flask, and incubate in a CO2 incubator.

**\*Caution:\*** When taking frozen cells from liquid nitrogen, if liquid nitrogen enters the cryovial, loosen the cap to release internal pressure, then quickly tighten and place on dry ice before placing in 37°C water bath to avoid explosion due to rapid nitrogen evaporation.

#### Cell Passage

1. Pass cells when confluency reaches over 85%.
2. In the biosafety cabinet, open the culture flask cap and collect the culture medium.
3. Add 3 ml sterile 1×PBS, lay flat to rinse cells, discard PBS.
4. Add 1 ml digestive solution to bottle, incubate at 37°C CO2 incubator for 1-2 minutes.
5. Observe under inverted microscope if cells have detached; if fully digested, add 2 ml complete medium with 10% FBS, and aspirate into a 15 ml centrifuge tube.
6. (If cells are not fully digested, perform additional digestion steps in portions using specific steps described.)
7. Centrifuge at 1000 rpm for 5 min.
8. Prepare two new T-25 flasks, add 4 ml complete medium to each.
9. After centrifugation, discard supernatant, resuspend pellet in 2 ml complete medium, split evenly into two T-25 flasks (1 ml each).
10. Place flasks in a 37°C incubator with 5% CO2 for static culture.

#### Cell Freezing

1. Follow steps 1-6 from the passage procedure.
2. After centrifugation and discarding supernatant, resuspend cells in 1 ml freezing medium to precipitate cells.
3. Transfer to 1.8 ml cryovials.
4. Place cryovials filled with DMSO in a programmable freezing container, then transfer to -80°C freezer overnight.
5. On the second day, transfer cryovials from freezing container into liquid nitrogen for long-term storage.

#### Testing NO production in trabecular meshwork cell lines:

###### I. Preparation of Working Solution

Take an appropriate amount of the stock solution and dilute it to the working concentration using the dilution buffer provided in this kit or self-prepared fresh culture medium (without serum and phenol red). For example,
take 5 µL of DAF-FM DA (5 mM) and dilute it at a 1:1000 ratio to prepare 5 mL of DAF-FM DA (5 µM) working solution. Mix thoroughly.

[Note] The working solution must be prepared fresh for immediate use. Discard any excess probe after the experiment. Since the dye is more prone to oxidation and decomposition in aqueous solution, please use it as quickly as possible.

[Note] The optimal working concentration of the probe needs to be determined based on your specific cell type or experimental conditions, or by consulting relevant literature. Generally, choose the minimum probe concentration that ensures sufficient fluorescence signal, as this helps minimize the accumulation of ester hydrolysis byproducts such as formaldehyde and acetic acid.

###### II. Probe Loading

* For cells with short stimulation times (usually within 2 hours): load the probe first, then stimulate the cells with an appropriate positive control and your drug of interest.
* For cells with long stimulation times (usually more than 6 hours): stimulate the cells with an appropriate positive control and your drug of interest first, then load the probe.

1. In-situ Probe Loading (Applicable only to adherent cells)

   1. Remove the cell culture medium and add an appropriate volume of the prepared DAF-FM DA working solution (e.g., the working solution diluted 1:1000 times). The volume added should be sufficient to cover the cells adequately; typically, add 1 mL of working solution per well of a 6-well plate.
   2. Incubate at 37°C for 20 minutes.
   3. Wash the cells three times with PBS, pH 7.4, to thoroughly remove any DAF-FM DA that has not entered the cells.
2. Probe Loading After Cell Harvesting

   1. After harvesting the cells, resuspend them in an appropriate volume of the prepared DAF-FM DA working solution (e.g., the working solution diluted 1:1000 times) to achieve a cell concentration of 1-20 million cells per milliliter.
   2. Incubate at 37°C for 20 minutes. The above operations can be performed in a centrifuge tube. Invert and mix gently every 3-5 minutes to ensure full contact between the probe and cells.
   3. Wash the cells three times with PBS, pH 7.4, to thoroughly remove any DAF-FM DA that has not entered the cells.
   4. Directly stimulate the cells with an appropriate positive control or your drug of interest, or first divide the cells equally into several aliquots and then stimulate them.

###### III. Fluorescence Detection

1. For samples with in-situ loaded probe: You can observe directly using a confocal laser scanning microscope (observation with a standard fluorescence microscope yields relatively poorer results), or harvest the cells and detect using a fluorescence spectrophotometer, fluorescence microplate reader, or flow cytometer.
2. For samples where the probe was loaded after cell harvesting: You can detect using a fluorescence spectrophotometer, fluorescence microplate reader, or flow cytometer. Direct observation with a confocal laser scanning microscope is also possible.
3. Parameter settings: Use an excitation wavelength of 495 nm and an emission wavelength of 515 nm. Perform real-time, time-lapse, or single time-point detection of fluorescence intensity before and after stimulation. The fluorescence spectrum of the DAF-FM-NO reaction product is very similar to that of Fluorescein. You can use the parameter settings for detecting Fluorescein, or alternatively, use the parameter settings for detecting FITC.

###### IV. Additional Notes

1. The recommended working concentration for DAF-FM DA in the above steps is 5 µM. For some cell types, if the fluorescence of the unstimulated negative control cells is also relatively strong, you can dilute DAF-FM DA at a ratio of 1:2000 to 1:5000, so that the working concentration of DAF-FM DA during probe loading is 1-2.5 µM. Conversely, if the fluorescence is weak after stimulation with your drug of interest, you can adjust the working concentration of DAF-FM DA to 10 µM to improve detection sensitivity.
2. The probe loading time can be adjusted appropriately within the range of 15-60 minutes based on the specific situation.

#### Testing cell apoptosis rate in retinal ganglion cell lines

1. Induce cell apoptosis using the experimental protocol.
2. Collect the cells

   * Suspension cells: Centrifuge at 300 × g for 5 min and aspirate the culture medium supernatant.
   * Adherent cells: Trypsinize the cells, preferably using trypsin without EDTA. Centrifuge at 300 × g for 5 min and aspirate the supernatant.
3. Wash the cells with ice-cold PBS and collect 1-5 × 105 cells.
4. Resuspend the cells in 500 µL of 1X Binding Buffer.
5. Add 5 µL of Annexin V-FITC and 5 µL of Propidium Iodide. Gently mix and incubate at room temperature for 10-20 minutes, protected from light.
6. Perform detection immediately after the reaction using a flow cytometer or fluorescence microscope. If immediate detection is not possible, keep the samples on ice and analyze within 1 hour.

   1. Flow Cytometry Detection:

      * Annexin V-FITC green fluorescence has an emission wavelength of 530 nm and can be detected using the FL1 (FITC detector) channel.
      * PI red fluorescence has an emission at 620 nm and can be detected using the FL2 or FL3 channel. Using the FL3 channel is recommended.
   2. Fluorescence Microscopy Observation:

      * Place a 30-50 µL drop of the cell suspension from Step 5 onto a glass slide and cover it with a coverslip. Observe under a fluorescence/confocal laser scanning microscope.
      * Cells positive for Annexin V-FITC staining (FITC filter, blue light excitation) will show bright green fluorescence on the cell membrane.
      * Cells positive for Propidium Iodide staining (PI filter, green light excitation) will show red fluorescence.
      * Early apoptotic cells are not stained by PI, while necrotic or late apoptotic cells show red cytoplasm, red nuclei, and a surrounding green cell membrane. Membrane shrinkage and blebbing can also be observed in late apoptotic cells.

▼

## Material and Method

### Plasmid purification kit

**Material:**

|  |  |
| --- | --- |
| RNase A | 60 μL |
| Buffer P1 | 60mL |
| Buffer P2 | 60mL |
| Buffer P3 | 80mL |
| Buffer W1 | 75mL |
| Elution Buffer | 25mL |
| EC column | 200 |

![Team Photo](https://static.igem.wiki/teams/5641/master/wetlab/plasmid-purification.webp)

A Plasmid Purification Kit is a critical resource in molecular biology, designed to isolate and purify plasmid DNA from bacterial cells, particularly after cloning procedures. In your project, using DH5α competent cells to replicate plasmids allows for the amplification of the desired DNA sequence. Once the bacteria have been cultured and the plasmids have been replicated, the purification kit facilitates the extraction of plasmid DNA, ensuring it is free from cellular debris and contaminants.

The typical workflow involves lysing the bacterial cells to release the plasmid DNA, followed by a series of purification steps. The kit contains specific buffers for cell lysis, neutralization, and washing, along with spin columns that selectively bind DNA. After lysis, the mixture is processed to remove proteins, RNA, and other cellular components, leaving behind high-quality plasmid DNA.Purified plasmid DNA is essential for various downstream applications, such as gel electrophoresis. The use of a plasmid purification kit not only ensures high yield and purity but also streamlines the preparation process, making it easier to obtain reliable results in experiments.

### Gel extraction kit

**Material:**

|  |  |
| --- | --- |
| Buffer GN (yellow) | 110mL |
| Buffer W1 | 75mL |
| Elution Buffer | 25mL |
| EC column | 200 |

![Team Photo](https://static.igem.wiki/teams/5641/master/wetlab/gel-extraction.webp)

A Gel Extraction Kit is a specialized tool used in molecular biology to recover DNA fragments from agarose gels following gel electrophoresis. After electrophoresis, when DNA samples are separated based on size, we often need to isolate specific bands of interest. The gel extraction process facilitates the extraction of these DNA fragments by utilizing a series of chemical and physical methods. After cutting out the desired gel slice containing the target DNA, the gel piece is treated with a binding buffer that helps solubilize the DNA and allows it to adhere to the resin in the spin column. Following this, the column is washed to remove impurities and contaminants, ensuring high purity of the extracted DNA. Finally, an elution buffer is used to release the purified DNA from the column. Gel extraction kits are essential for various downstream applications, such as cloning, sequencing, and PCR amplification, making them an invaluable resource in genetic research and molecular diagnostics. Their convenience and efficiency streamline the process of obtaining high-quality DNA for further experimentation.

### Protein purification kit

**Material:**

|  |  |
| --- | --- |
| His-tag Purification Resin (Reductant and Chelator Resistant) | 10 mL |
| Native Lysis Buffer | 120mL |
| Native Wash Buffer | 60mL |
| Native Elution Buffer | 60mL |
| Lysozyme | 60mg |
| Empty Affinity Chromatography Column (3 mL) | 10 sets |
| DTT (solid) | 10X 1 tube |

![Team Photo](https://static.igem.wiki/teams/5641/master/rot/his-tag-protein-purification-kit-rot.webp)

A Protein Purification Kit is an essential tool in biochemistry and molecular biology, designed to isolate and purify specific proteins from complex mixtures, such as cell lysates or tissue extracts. In our experiments, we use BL-21 as a medium to produce protein. The primary function of these kits is to enable us to obtain high-purity proteins for various applications, including structural studies, functional assays, and therapeutic development. The purification process often involves several techniques, including affinity chromatography, ion-exchange chromatography, and size-exclusion chromatography. These methods exploit differences in protein size, charge, and binding affinity, allowing for a systematic approach to separate the desired protein from contaminants. Additionally, protein purification kits are designed to be user-friendly, providing detailed protocols and optimized conditions to streamline the purification process. This ease of use, combined with the ability to achieve high yields and purity levels, contributes to more accurate results in our iGEM journey.

### ELISA kit

**Material:**

|  |  |
| --- | --- |
| High Standard | 2 vial |
| Reconstitution Solution | 2 vial |
| Standard & Sample Diluent | 25mL |
| Microelisa Stripplate | 96T / 48T |
| Bio-Antibody | 10mL |
| HRP-Conjugate Reagent | 10mL |
| TMB | 10mL |
| Stop Solution | 6 mL |
| 20X Wash solution | 25mL |

![Team Photo](https://static.igem.wiki/teams/5641/master/wetlab/elisa-1.webp)

![Team Photo](https://static.igem.wiki/teams/5641/master/wetlab/elisa-2.webp)

![Team Photo](https://static.igem.wiki/teams/5641/master/wetlab/elisa-3.webp)

![Team Photo](https://static.igem.wiki/teams/5641/master/wetlab/elisa-4.webp)

![Team Photo](https://static.igem.wiki/teams/5641/master/wetlab/elisa-5.webp)

An ELISA (Enzyme-Linked Immunosorbent Assay) Kit is a powerful tool used to quantify and analyze the binding affinity of receptors and protein in a sample. In our project, using the ELISA kit allowed for the assessment of optimal dosages and the binding interactions between specific biomolecules. The process typically involves immobilizing an antigen or antibody on a microplate, followed by adding samples that may contain the target molecules. After incubation, a secondary enzyme-linked antibody is introduced, which binds to the target, allowing for the detection of bound complexes.In our experiment, we tested five sets of ELISA kits which enabled a comprehensive analysis of different concentrations and conditions, helping to establish a standard dosage response curve. After performing the assay, the optical density (OD) of each well is measured, providing quantitative data on the binding affinity. This data can then be plotted to visualize the relationship between dosage and binding activity.The resulting graph aids in determining the optimal dosage for further experiments and provides insights into the efficiency and specificity of the interactions being studied. Overall, the ELISA kit is invaluable in research, diagnostics, and therapeutic development, allowing for precise measurement of biomolecular interactions and guiding experimental design.

### DAF-FM DA

**Material:**

|  |  |
| --- | --- |
| DAF-FM DA(6mM in DMSO) | 20 µL |
| Dilution Buffer | 50 mL |

![Team Photo](https://static.igem.wiki/teams/5641/master/wetlab/daf-fm-da.webp)

A DAF-FM DA Kit is a specialized tool used in cell biology to detect intracellular nitric oxide (NO) release, which plays a crucial role in various physiological and pathological processes, including apoptosis. The kit utilizes DAF-FM DA (4-amino-5-methylamino-2',7'-difluorofluorescein diacetate), a cell-permeable fluorescent probe that enters living cells and is hydrolyzed by intracellular esterases to DAF-FM, a compound that reacts specifically with NO to form a highly fluorescent benzotriazole derivative. This fluorescence can be detected and quantified by fluorescence microscopy or flow cytometry, providing a sensitive and convenient method for measuring real-time NO production within cells. The kit typically includes DAF-FM DA probe and buffer solutions for staining cells. Cells are incubated with DAF-FM DA, allowing the probe to diffuse inward and become activated, and the fluorescence intensity reflects the intracellular NO levels. The DAF-FM DA Kit is an invaluable resource for studying NO-related signaling pathways, oxidative stress responses, and apoptosis mechanisms in various cellular models. This detection method is commonly applied in research investigating the role of NO in cell death and other biological functions.

### Annexin V-FITC/PI Apoptosis Kit

**Material:**

|  |  |
| --- | --- |
| Annexin V-FITC | 100 µL |
| 1X Binding Buffer | 10 mL |
| Propidium Iodide | 100 µL |

![Team Photo](https://static.igem.wiki/teams/5641/master/wetlab/annexin-apoptosis-kit.webp)

An Annexin V-FITC/PI Apoptosis Kit is a specialized tool used in cell biology to detect and distinguish apoptotic cells at different stages. The kit employs Annexin V, a Ca2+-dependent phospholipid-binding protein that selectively binds to phosphatidylserine (PS), which normally resides on the inner leaflet of the cell membrane but translocates to the outer surface in early apoptosis. Annexin V is conjugated to the fluorescent dye FITC, allowing apoptotic cells with exposed PS to be identified by flow cytometry or fluorescence microscopy. Additionally, the kit includes Propidium Iodide (PI), a nucleic acid dye that cannot penetrate intact cell membranes but enters cells with compromised membranes, such as late apoptotic or necrotic cells, binding to DNA and emitting red fluorescence. By combining Annexin V-FITC and PI staining, researchers can distinguish live cells (Annexin V-/PI-), early apoptotic cells (Annexin V+/PI-), and late apoptotic or necrotic cells (Annexin V+/PI+). This dual staining method provides a sensitive, rapid, and convenient approach for assessing apoptosis levels, making the kit essential in apoptosis research, drug screening, and cell viability studies. The kit typically includes FITC-labeled Annexin V, PI solution, and a binding buffer, with staining completed in about 10 to 40 minutes for analysis by flow cytometry or microscopy. This method contributes to understanding cellular mechanisms, disease pathology, and therapeutic interventions by providing precise apoptosis detection in cell samples.

### Protein Refolding Kit

**Material:**

|  |  |
| --- | --- |
| Buffer 1- buffer 15 | 950µL x 10 |
| DTT | 1mL x10 |
| glutathione, reduced | 1mL x 10 |
| glutathione, oxidized | 1mL x 10 |
| Lysis buffer | 50nM Tri-HCL pH7.5, 0.1mM NaCl, 0.1nM EDTA, 0.1mM DTT ,5% glycerol |

![Team Photo](https://static.igem.wiki/teams/5641/master/wetlab/protein-refolding.webp)

A Protein Refolding Kit is a crucial resource in protein engineering, especially when working with recombinant proteins expressed in E. coli, which often form inclusion bodies. In our project, these inclusion bodies can trap the target protein, leading to misfolding or unfolding due to the presence of a membrane that encapsulates them. The refolding kit provides a systematic approach to recover functional proteins by breaking down this membrane and facilitating the refolding process. With 15 different pH buffers included in the kit, we can systematically test various conditions to identify the optimal pH that promotes proper refolding of your protein. This flexibility allows for fine-tuning the refolding environment, enhancing the chances of achieving the correct three-dimensional structure necessary for the protein's biological activity. By utilizing the protein refolding kit, we can effectively recover and purify the functional protein, paving the way for further studies and applications in our research.

### BCA Kit

**Material:**

|  |  |
| --- | --- |
| BCA reagent | 100mL |
| Cu reagent | 3mL |
| PBS diluent | 30mL |
| BSA protein standard (5 mg/mL BSA) | 1mL |

![Team Photo](https://static.igem.wiki/teams/5641/master/wetlab/bca.webp)

The primary function of the BCA (Bicinchoninic Acid) Test Kit is to measure the concentration of protein in a sample. By quantifying the amount of protein, researchers can assess the purity and yield of their protein preparations, monitor protein expression levels, and ensure proper concentrations for various experimental applications. The assay provides a reliable and efficient method for protein analysis, making it an essential tool in many areas of biological research.

## Safety

### Overview

Safety is of top priority to us at our lab. We follow stringent procedures to guarantee a clean environment and reduce risks when conducting tests. Our team is routinely trained in emergency protocols and best practices. Only authorized personnel with permission can enter sensitive locations, and wearing personal protection equipment is required. Routine inspections help us identify probable hazards, nurturing a culture of proper safety awareness thus allowing us to focus on delivering reliable and accurate research results.

### Laboratory Training

Before commencing any experimental procedures, it is imperative that every team member is thoroughly familiar with the laboratory setup, personal protective equipment (PPE), crisis management protocols, biosafety measures, and established laboratory rules. To achieve this, multiple sessions of wet lab training are conducted to equip each member with the necessary skills to effectively handle various situations that may arise in the lab. Participants are educated on the critical importance of maintaining a safe working environment and the specific regulations that govern laboratory conduct. A substantial emphasis is placed on the safe handling and disposal of biohazardous materials, including organisms such as \*Escherichia coli\* and waste items like agar plates. Furthermore, we underscore the necessity of wearing PPE, including gloves and lab coats, to prevent contamination and mitigate the risk of chemical exposure. Fire safety also constitutes a vital aspect of our training; team members receive instruction on the location and proper use of fire extinguishers, as well as the appropriate responses to fire emergencies. By providing comprehensive knowledge of potential risks and safety measures, we empower all team members to recognize hazards and adopt best practices that enhance safety within the laboratory environment.

#### Cloning

In our experiments, we utilize NEB®️ Stable Competent E. coli (BL21 and DH5α) as our chassis. The classification of these microorganisms as Risk Group 1 signifies their non-pathogenic nature, ensuring their safety for application in educational environments, such as secondary school laboratories. Nonetheless, to ensure safety when handling these procedures, personnel must wear gloves while conducting experiments involving these strains.

![Team Photo](https://static.igem.wiki/teams/5641/master/edu/ww/screenshot-2025-10-07-194638.webp)

#### SDS-PAGE

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) is employed in our project to analyze the binding interactions between each strand and the target RNA oligos. Proper safety precautions are taken because the acrylamide used in this technique is a neurotoxin. Personnel must wear gloves and masks when handling the acrylamide solution and gels. We use TEMED during the gel manufacturing process, which poses the risk of digestive and respiratory tract burns. Hence, we use pre-cast gels to diminish these risks and foster a safer working environment.

![Team Photo](https://static.igem.wiki/teams/5641/master/rot/sds-buffer-rot.webp)

#### Agarose Gel

Ethidium bromide, commonly used for DNA staining, is recognized as a carcinogen. To prioritize the safety of our laboratory personnel, we adopt SYBR Safe as a safer substitute for post-staining agarose gels. This decision preserves effective DNA visualization while drastically lowering health risks.

#### Lab Safety Measures

To ensure a safe working environment in our laboratory, following critical safety measures are strictly enforced:

1. Personal Protective Equipment: Gloves are vital and compulsory when handling bacteria and using hazardous chemicals to protect personnel from exposure.

   ![Team Photo](https://static.igem.wiki/teams/5641/master/wetlab/gloves.webp)
2. Clear Labeling: All containers involving hazardous chemicals and biological materials are properly labelled, making identification simple and guaranteeing correct handling.

   ![Team Photo](https://static.igem.wiki/teams/5641/master/rot/screenshot-2025-10-05-011207.webp)
3. Biosafety cabinet Usage: Hazardous chemical experiments are carried out in fume hoods to reduce the risk of inhalation and ensure proper ventilation.

   ![Team Photo](https://static.igem.wiki/teams/5641/master/rot/screenshot-2025-10-05-011655.webp)
4. Prohibition of Food and Drink: Eating and drinking in the laboratory are strictly forbidden to prevent any potential contamination of samples and work areas.
5. Hand Sanitization: To ensure personal safety and stop the release of contaminants outside of designated locations, all laboratory personnel must disinfect with alcohol and thoroughly wash their hands after finishing work.

   ![Team Photo](https://static.igem.wiki/teams/5641/master/wetlab/handsanitizer.webp)
6. Regular Sanitation: Workbenches are sterilized with alcohol frequently to maintain cleanliness, ensure asepsis, and lessen the chances of contamination.
7. Waste Disposal: To ensure proper waste management, laboratory waste must be disinfected using a diluted bleach solution (1:49) before being disposed of.

   ![Team Photo](https://static.igem.wiki/teams/5641/master/wetlab/wastedisposal.webp)
8. Supervision: To guarantee complete compliance with safety regulations and protocols, every experiment is carried out under the guidance of a certified educator.
9. Safety Information and Emergency Response: We have a corner detailing fire escape routes, safety rules, first aid kit and fire extinguishers. Additionally, all wet lab members are briefed on laboratory rules, potential hazards, and emergency procedures.

   ![Team Photo](https://static.igem.wiki/teams/5641/master/wetlab/firstaidcorner.webp)

Our ultimate goal is to create a safe and healthy environment for everyone involved in our experiments, and we are committed to upholding these standards rigorously.

{% endblock %}

{% extends "layout.html" %}
{% block page\_content %}

![IMG](https://static.igem.wiki/teams/5641/master/homepage/project.webp)

# RESULTS

### Results

* [Introduction](#section1)
* [Cycle 1](#section2)
* [Cycle 2](#section3)
* [Summary of key findings](#section4)

## Introduction

Throughout this project, our team has conducted several key tests, including gel electrophoresis, SDS-PAGE, BCA test (only in cycle 2), and ELISA, each serving an important role in analyzing and verifying our proteins. In the two cycles, we initially carried out gel electrophoresis to confirm the incorporation of the designed gene into the bacterial hosts. Upon obtaining successful results from gel electrophoresis, we proceeded with plasmid transformation into the bacteria to facilitate protein expression. The transformed bacteria were then grown through cell culture to express the target protein. After that, a BCA test was conducted to find out the initial concentration of our samples. Subsequently, SDS-PAGE was conducted to verify the expression of the target protein. Finally, ELISA was employed to evaluate the binding affinity of the fusion peptides. Below are the successful results from these experiments.

## Cycle 1

In the first cycle, we designed a fusion protein utilizing PEP-1 and FWT, which consists of FRATtide, Wnt3α,
and TP1. Additionally, we incorporated another fusion peptide known as BC, which consists of BDNF and CDR1. For
the expression of our genes and proteins, we have used DH5-α and BL21 strains.

### GEL ELECTROPHORESIS

We used gel electrophoresis for separating proteins, DNA, and RNA based on their size and charge. It operates
on the principle of applying an electric field to move charged molecules through a gel matrix. Molecules
separate based primarily on size and charge, producing distinctive bands. Successful results are indicated by
clear, well-resolved bands that correspond to expected sizes, while smearing, distortion, or unexpected bands
suggest experimental failure, degradation, or contamination.

To confirm that the bacteria contain our designed gene, we used gel electrophoresis to verify the presence of a
band matching the expected size of our gene.

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/fwtgel.webp)

Fig 1 FWT Gel electrophoresis

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/bcgel-1.webp)

Fig 2 BC Gel electrophoresis

#### Result

In the pictures above, we can see the FWT band positioned around 1500 bps, while the BC band is at around 1000
bps. Both of them match our estimated base pair lengths, leading to the conclusion that our design was
functioning as intended.

### Transformation and cell culture

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/okok/screenshot-2025-10-08-at-17-11-12.avif)

Fig 3 Successful transformation of FWT, BC to DH5-α

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/okok/screenshot-2025-10-08-at-17-11-00.avif)

Fig 4 Successful transformation of FWT, BC to BL21

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/fwt-bc-culture.webp)

Fig 5 Successful culture of FWT, BC

#### Result

In the pictures above, we have done transformation of BC, FWT to BL21 and DH5-α. After the transformation success, we have cultured the cells with our plasmid.

### SDS-PAGE

SDS-PAGE is a technique commonly used in synthetic biology experiments to separate proteins based on their
molecular weight. In this process, proteins are denatured and uniformly coated with negative charges,
allowing for separation based purely on size. After denaturation, the proteins are loaded onto a polyacrylamide
gel and subjected to an electric field. The proteins migrate through the gel, with smaller proteins moving
faster than larger ones. The success of the technique is evaluated by the presence of sharp, distinct bands at
the expected molecular weights. If issues arise, troubleshooting typically focuses on the shape of the bands,
the resolution of the gel, or the integrity of the gel itself.

In our project, SDS-PAGE was used to confirm the presence of our targeted proteins by verifying their expected
molecular weights on the gel.

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/okok/sds-new.avif)

Fig 6 SDS-PAGE results of FWT and BC

#### Result

In the first cycle, the results were successful and aligned with our expectations, showing a clear and linear result. Both the FWT and BC lanes matched our estimated kDa, which is approximately 35 kDa. This outcome verifies the presence of our protein, confirming that both transformations of FWT and BC are successful. With these results, we can proceed to the next steps of our testing, which include conducting an ELISA kit.

### ELISA

ELISA kits quantify specific proteins or antigens by using antibody binding and enzymatic colorimetric
reactions. The sample antigen binds to a capture antibody on a microplate, then a detection antibody linked to
an enzyme is added. Substrate addition produces a color change proportional to antigen concentration. Successful
assays produce clear, reproducible signals with controls ensuring specificity and sensitivity. Data are analyzed
using standard curves to determine precise protein concentrations, with options for multiplexing or kinetic
measurements for enhanced analysis.

#### Result

In our initial ELISA assay, a standard curve quality of an ideal R² value was obtained in the BC component,
confirming the structural integrity and functional efficacy of our DNA construct. For the color intensity, the
BC component exhibited a strong yellow signal, indicating substantial protein expression and providing a solid
foundation for its further development. In contrast, the absence of a yellow signal and detectable absorbance
for the FWT component suggests a lack of protein production.

## Cycle 2

In the second cycle, we began by replacing FWT with FT while keeping BC unchanged. Additionally, we substituted
BL21 cells with SHuffle cells to enhance protein expression. The tests performed in cycle 1, including SDS-PAGE,
gel electrophoresis, and ELISA, were repeated to test our newly designed plasmid.

content

### Gel electrophoresis

Gel electrophoresis was conducted, and the results indicated the success of our DNA expression in bacterial
cells with the newly designed plasmid.

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/gel-2.webp)

Fig 7 BC and FT Gel electrophoresis

#### Result

Similar to cycle 1, we confirmed the presence of our targeted gene by conducting gel electrophoresis. The results of the experiment were consistent with our expected bps, which are around 1000 bps, confirming the presence of FT, BC, and other separated proteins’ DNA and indicating that both transformations for BC and FT were successful. This enables us to move forward with additional tests.

### Transformation and cell culture

![Team Photo](https://static.igem.wiki/teams/5641/master/edu/ww/dday/screenshot-2025-10-08-at-16-14-52.webp)

Fig 8 Successful transformation of FT, BC to DH5-α

![Team Photo](https://static.igem.wiki/teams/5641/master/edu/ww/dday/screenshot-2025-10-08-at-16-15-28.webp)

Fig 9 Successful transformation of FT, BC to SHuffle cells

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/ft-bc-culture.webp)

Fig 10 Successful culture of FT, BC

### SDS-PAGE

SDS-PAGE was conducted to check the existence of our protein.

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/sds-2.webp)

Fig 11 SDS-PAGE results of FT and BC

#### Result

In the second cycle, we verified the presence of our recombinant plasmid using SDS-PAGE analysis. The results aligned with our expectations, showing bands in all lanes at approximately 35 kDa—our predicted molecular weight—verifying the existence of our protein once again and ensuring that both transformations for BC and FT succeeded. Moreover, the protein expression levels in SHuffle cell cultures were higher than those in BL21, confirming our hypothesis. These successful outcomes enabled us to proceed with further tests.

### BCA

The Bicinchoninic Acid (BCA) protein assay works by first reducing copper ions (Cu2+) to cuprous ions (Cu1+) in an alkaline environment, a reaction facilitated by the peptide bonds and certain amino acid residues like cysteine, tyrosine, and tryptophan in the protein sample. The cuprous ions then react with bicinchoninic acid to form a purple-colored complex that strongly absorbs light at 562 nm. The intensity of this color change is directly proportional to the protein concentration in the sample. By measuring the absorbance of the purple complex using a spectrophotometer and comparing it to a standard curve made from known protein concentrations, the total protein content can be accurately quantified. The assay is typically incubated at 37 to 60 °C to enhance sensitivity and achieve consistent results.

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/bca-graph.webp)

Fig 12 standard curve of BCA

![Team Photo](https://static.igem.wiki/teams/5641/master/wetlab/bca1.webp)

Fig 13 Results of BCA

#### Result

We have conducted a BCA test. The R² value of the standard curve is 0.996. The concentrations of our refolded protein has been discovered, which is 0.42121478 mg/mL

### ELISA

After obtaining successful results with SDS-PAGE and gel electrophoresis, we proceeded to ELISA tests to
evaluate the binding affinity of the fusion peptides.
We have put the ELISA kit assays up for an absorbance test, the results are shown below.

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/elisa-anp32a.webp)

Fig 14 Elisa results of ANP32A

![Team Photo](https://static.igem.wiki/teams/5641/master/edu/ww/dday/anp32a.avif)

Fig 15 ELISA graph of ANP32A

![Team Photo](https://static.igem.wiki/teams/5641/master/edu/ww/dday/image-1.avif)

Fig 16 Elisa results of TrkB, PI3k, NOS, GSK3.

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/elisatrkb2.avif)

Fig 17 ELISA graph of TrkB.

![Team Photo](https://static.igem.wiki/teams/5641/master/engineering/elisapi3k.webp)

Fig 18 ELISA graph of PI3K.

![Team Photo](https://static.igem.wiki/teams/5641/master/edu/ww/dday/nos.avif)

Fig 19 ELISA graph of NOS.

![Team Photo](https://static.igem.wiki/teams/5641/master/edu/ww/dday/gsk3.avif)

Fig 20 ELISA graph of GSK3.

#### Result

The data indicate a substantial presence of protein in BC, as evidenced by the prominent yellow coloration observed in the result images. The results and the calibration curve allows us to find out binding affinities and optimal dosage of our peptides with targeted receptors. In addition to the visual confirmation, the quality of the standard curve is optimal. Specifically, the R² value for PI3K was measured at 0.9937, for ANP32A at 0.9935, and for TrkB at 0.9975, which collectively support the robust use of BC in these experiments. Also, under 0.12062 mg/mL of BC protein concentration, the binding concentration of PI3K was at 1.899175069 ng/mL , ANP32A at 0.151433472 ng/mL, and TrkB at 0.058789625 ng/mL.

The findings for FT were consistent with those for BC. Strong yellow tones appeared in the FT result images, suggesting a notable protein presence as well. Furthermore, the standard curves achieved in FT tests were of excellent quality. For PI3K, the R² value reached 0.9718, for NOS it was 0.991, and for GSK3 the value stood at 0.9972, indicating FT had successful results in all three experiments. Under 0.12062 mg/mL of FT protein concentration, the binding concentration of PI3K was at 1.938588 ng/mL ,NOS at 0.592933444 µmol/mL, GSK3 at 12.78571429 pmol/L.

## Summary of key findings

1. The BC fusion peptide showed consistently positive results across gel electrophoresis, SDS-PAGE, and ELISA
   assays, confirming its effective expression and functional activity.
2. ELISA tests on the FWT protein revealed no measurable color development and had a poor standard curve quality, indicating insufficient protein expression and a lack of functionality.
3. The redesigned FT protein demonstrated successful expression and functionality in all conducted assays,
   confirming the improved performance of the modified construct.

Based on these findings, we critically evaluated our initial approach and made strategic modifications to
optimize the project’s outcomes. We shifted our focus toward improving protein design and expression. This
iterative process allowed us to address initial shortcomings and refine our experimental strategy for better
performance and reliability.
For more comprehensive information about our project, please visit our [engineering page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D), which provides a detailed overview of
designing, building and testing of our eye drop.

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