iGEM 2024 — Team links-china

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June to July Notebook](#d1)
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August Notebook](#d2)
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September Notebook](#d3)

June to July Notebook

August Notebook

September Notebook

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Overview](#d1)
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DBTL Cycle I: Characterization of Binding Domains](#d2)
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DBTL Cycle II: Characterization of Defensins](#d3)
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DBTL Cycle III: Fusion Expression of Defensins](#d4)
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DBTL Cycle IV: Universality of Antimicrobial Binding Domains](#d5)
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DBTL Cycle V: Application](#d6)

Overview

As outlined in our [project design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D), we aim to verify both the binding activity of our binding domain to the target material and the antimicrobial efficacy of our functional domain, defensins. The overall project workflow is illustrated in Fig. 0. Our approach involves screeninng and validating the binding and functional domains separately before combining them. To screen and validate the binding domain, we linked it to a chromoprotein, enabling visual confirmation of its successful attachment to the target material. This fusion of the binding domain and chromoprotein is referred to as BDC. For the functional domain, we first expressed defensin independently then expressed SUMO-linked defensin for enhanced stability. Ultimately, our goal is to fuse both domains into a single protein, where the binding domain is connected to SUMO, which is in turn linked to the functional domain. This fusion protein is designed to impart antimicrobial properties to first-aid materials.

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Fig.0 Simplified outline of our project

DBTL Cycle I: Characterization of Binding Domains

Construction of binding domain-chromoprotein expression strain

In Cycle I, our goal is to obtain a fusion protein composed of a binding domain linked to a chromoprotein—namely mTurquoise, sfGFP, fwYellow, gfasPurple, mRFP, and eforRed—to enable visualization of the ability of the binding domains—specifically CBM2, CBM3, CBM5, CBMxx, α1, and α2; respectively—to integrate with the target material.

We performed codon optimization of CBM2, mTurquoise, α1, CBMxx, and gfasPurple, obtaining them through DNA synthesis. The fragments CBM3, sfGFP, fwYellow, mRFP, α2, and eforRed were obtained via PCR amplification using the primers Alyssa-F1/R1, Qiao-F/R, Carl-F/R, Arendelle-F/R, Hugo-F2/R2, and Hugo-F1/R1 from vector A038, vector pKeystone012, vector J23102-11 fwYellow, vector pSEVA321-rhaR-rhaS-RiboJ-RFP, pET28a-α2β1-HNP1, and pKeystone009, respectively. We obtained the vectors A038 from iGEM Team LINKS China 2021; pKeystone012 and pKeystone009 from iGEM Team Keystone 2023; and J23102-11 fwYellow from LinkSpider Lab (Shenzhen, China). The DNA fragment encoding CBM5 was obtained through SOE PCR using the primer pairs CBM5MMP-F1/R1, CBM5MMP-F2/R2, and CBM5MMP-F3/R3. The primer sequences are shown in [experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D).

All final binding domain DNA fragments and chromoprotein DNA fragments, except for α2 and eforRed, were ligated into the digested plasmid pLINKS2400 expression vector by Golden Gate assembly; α2 and eforRed were assembled into pLINKS2400 through Gibson assembly. *Escherichia coli* DH5a competent cells were transformed and screened in LB medium containing kanamycin. The plasmids were then confirmed by colony PCR and DNA sequencing for the presence of the correct sequence. The correct plasmids were named pLINKS2407, pLINKS2408, pLINKS2410, pLINKS2405, pLINKS2406 and pLINKS2431 for plasmids carrying CBM2-mTurquoise, CBM3-sfGFP, CBM5-fwYellow, α1-mRFP, α2-eforRed and CBMxx-gfasPurple, respectively. These plasmids were then extracted and transformed into *E. coli* BL21 (DE3) competent cells. pLINKS2431 is later transformed into SHuffle T7 cell for expression of CBMxx-gfasPurple in supernatant.

For optimal conditions, *E. coli* BL21 (DE3) and *E. coli* SHuffle T7 cells containing the correct plasmids pLINKS2407, pLINKS2408, pLINKS2410, pLINKS2405, pLINKS2406, and pLinks2431 were cultured overnight in fresh LB medium at 37 °C. Take 400 µL of the overnight culture and inoculate it into 400 mL of LB medium containing kanamycin. Incubate the culture at 37°C until OD-600 reaches 0.6-0.8. Then add IPTG with a final concentration of 0.3 mM and continue incubation at 30°C with shaking at 250 rpm for 3.5 hours. For α2-eforRed and CBMxx-gfasPurple fusion proteins, we observed suboptimal expression under these conditions. Thus, we modified the protocol by using a lower IPTG concentration of 0.1 mM and reducing the temperature to 20°C, followed by an extended induction period of 14 hours. After induction, the cells were collected by centrifugation at 4 °C and 8500 rpm for 3.5 minutes. The pellet was suspended in 40 ml of 20 mM Tris-HCl, pH 8.0, and sonicated on ice with an appropriate tip at 25 Hz for 30 minutes (3 seconds working, 6 seconds free) until the cells were lysed. The supernatant of the cell lysates was collected by centrifugation at 4 °C and 8,500 rpm for 10 minutes. SDS-PAGE was then performed to analyze correct protein expression, and fluorescence was detected under yellow-light to visualize protein expression.

![](https://static.igem.wiki/teams/5185/figures/eng/engineering-fig-1.png)

Figure 1

Verification of binding domain function

To test the binding domain protein fusion with cellulose, two rows of proteins, including two of the BDCs targeted to bind to cellulose (CBM2-mTurquoise and CBM3-sfGFP) and a fluorescent protein control (mTurquoise and sfGFP), were added to a 48-well plate, all suspended in 20mM Tris-HCl. Row 1 contained gauze soaked in the protein solution, while Row 2 contained the protein only. After soaking, washing with isosopranol and drying (more details in [experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)), the results indicate that CBM2 and CBM3 are capable of binding with cellulose, as shown by the brighter fluorescent after washing(Fig. 2A).

For BDCs intended to bind to collagen/chitosan and sodium alginate hydrogel mixture, we conducted procedures as outlined in the [experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D). It is important to note that the α1 and α2 binding domains are intended to interact specifically with type IV collagen. However, due to budget constraints in our lab, we assessed their binding ability using type II collagen instead. This choice may explain the comparatively lower brightness observed in the α binding domains incorporated into the sodium alginate and collagen mixture after washing. We opted to use α2 to verify the functionality of our combined domain (the binding domain fused with the functional domain) because of its relatively greater brightness. CBM5 and CBMxx, on the other hand, both displayed moderate ability to bind to their target materials as indicated by their relative deepness in color compared to the control group. Thus, we continued to use both binding domains to verify our combined domain functions.

![](https://static.igem.wiki/teams/5185/figures/eng/engineering-fig-2.png)

Figure 2 (a) Binding efficiencies of cellulose binding domains CBM2 and CBM3 toward cellulose gauze (b) Binding efficiencies of collagen binding domains α1 and α2; chitosan binding domain CBM5; alginate binding domain CBMxx toward respective hydrogel materials

DBTL Cycle II: Characterization of Defensins

After verifying the binding domain function, we next wanted to verify if the antimicrobial domain is functioning normally. Due to the extremely low content of defensins in human blood and the high cost of chemical synthesis, we used *Escherichia coli* to express the selected four human recombinant defensins (HNP1, HNP4, HD5 and HBD3). However, the prokaryotic expression of defensins is not ideal, either the expressed defensins lack function, or the leakage of expressed defensins kills the cells.

In addition, we also synthesized the immature forms of two defensins (preproHNP1 and preproHD5). Xie et al reported that the preproHNP1 expressed in BL21(DE3) can be cleaved by the endogenous serine protease in *E. coli* to produce the mature HNP1, so *E. coli* can accumulate more defensins after reaching a higher cell density.

Based on this, we replaced the chassis with SHuffle T7, so that the defensins expressed in *E. coli* can be correctly folded and form disulfide bonds, thereby improving the stability of the defensins.

Construction of defensin expression strains

Defensin fragments were obtained by SOE-PCR as described in the protocol. Those fragments were ligated into the digested plasmid pLINKS2400 expression vector by golden gate assembly. *Escherichia coli* DH5a competent cells were transformed and screened in LB medium containing kanamycin. The plasmids were then confirmed by colony PCR (figure 3a) and DNA sequencing for the presence of the correct sequence. The correct plasmids were named pLINKS2401, pLINKS2402, pLINKS2403, and pLINKS2404 for plasmids carrying HNP1, HNP4, HD5, and HBD3, respectively. These plasmids were then extracted and transformed into *E. coli* SHuffle T7 competent cells.

For optimal conditions, *E. coli* SHuffle T7 cells containing correct plasmid pLINKS2401 ~ pLINKS2404 were cultured in fresh LB medium for overnight at 37 °C. Next day, the overnight cultures were subcultured in 4 ml LB medium with Kanamycin on a rotary shaker (220 rpm) at 30 °C for 3.5 h until OD600 reached 0.6~0.8. For expression of the defensins, *E. coli* SHuffle T7 cells containing pLINKS2400 were then cultured in 300 ml LB medium at 37 °C for 3 h until OD600 reached 0.6 to 0.8, the expression of the gene was induced by addition of 0.3 mM IPTG and conducted for 3.5h at 30 °C. The cells were collected by centrifugation at 4 °C, 12000 rpm for 3 min. The pellet was suspended in 500 ul 20mM Tris-HCl，pH8.0 and sonicated in ice with an appropriate tip at 25 Hz for 4 min (3 s working, 6 s free) until cells were lysed. Tris-Tricine-SDS-PAGE was then performed to analyze the expression of proteins in the cell, as shown in Figure 3b. The result shows that for the defensins without the pro segment, only HNP1 were expressed, but only in small amounts. Moreover, we observed that these strains continued to rapidly increase in cell number after IPTG induction, which is contrary to the expected results. If the produced defensin is toxic to *E. coli*, the growth of cells would be rapidly inhibited. We speculate that there are two reasons why the defensins are not produced and is not toxic. One reason is that the molecular weight of the defensin is too small (less than 5 kDa), making it easily degraded by endogenous proteases. The second reason is that the addition of 10 amino acids at the N-terminus, including a 6×His tag, affects the correct folding of the defensins. Therefore, we have decided to change our strategy and use protease cleavage to release the defensins, and after N-terminal protease cleavage, it does not contain any amino acid residues.

![](https://static.igem.wiki/teams/5185/figures/eng/engineering-fig-3.png)

Figure 3 (a) Identification of expression vector pLINKS2401-2404. The target vectors are about 400 bp length, as indicated with arrows. (b) SDS-PAGE analysis of the recombinant defensins expression in*E. coli*SHuffle T7. The target proteins have a molecular weight about 3.5 kDa for HNPs and 11 kDa for PreHNPs before cleavage by protease, as indicated with arrows.

Meanwhile, ProHNP1 and ProHD5 were both produced, but had not been cleaved by protease naturally expressed in*E. coli*SHuffle T7 into mature form of defensins and thus didn’t inhibit the growth of E. coli.

DBTL Cycle III: Fusion Expression of Defensins

Due to the unsuccessful attempts to express defensins directly in *Escherichia coli*, we decided to produce defensins using an enzymatic cleavage method. The SUMO tag, known for enhancing solubility and leaving no amino acid residues at the N-terminus after cleavage, has been widely utilized in the production of antimicrobial peptides (AMPs) in previous studies (Zhu et al., 2021). Building on this, we fused the SUMO tag to the C-terminus of the functionally validated binding domain CBM3 and the N-terminus of the functionally unvalidated HNP1, aiming to generate defensins through enzymatic cleavage.

Construction of CBM3-SUMO-Defensins strain

We obtained CBM3! fragment through amplification of A038 using primers Alyssa-F1 and Alyssa-R1, and SUMO! from pET28a-BsaI site1-SUMO-BsaI site2 using primers Henry-F1 and Henry-R1. Defensin fragments were obtained via amplification of their corresponding vectors pLINKS2401 ~ pLINKS2404. The CBM3!, SUMO!, and defensin fragments were ligated into the digested plasmid pLINKS2400 expression vector via golden gate assembly. The process of transformation and confirmation is the same as above. The confirmed plasmids were named pLINKS2416, pLINKS2417, pLINKS2418, and pLINKS2419, corresponding to plasmids carrying the HNP1, HNP4, HD5, and HBD3 genes（fig. 4a）, respectively. These verified plasmids were then extracted and transformed into *E. coli* SHuffle T7 competent cells. The IPTG-induced expression of CBM3-SUMO-defensins was validated in the same manner as the defensins. SDS-PAGE analysis revealed the presence of the correct bands (Fig. 4b). Subsequently, we scaled up the fermentation to 400 mL. Once the optical density (OD) reached 0.6–0.8, we induced expression with 0.1 mM IPTG at 20°C for 12 hours. Afterward, we lysed the cells and purified the target protein from the cell lysate using a Ni-NTA affinity chromatography column.

![](https://static.igem.wiki/teams/5185/figures/eng/engineering-fig-4.png)

Figure 4 (a) Identification of expression vector pLINKS2416-2419. The target vectors are about 1200 bp length, as indicated with arrows. (b) SDS-PAGE analysis of the CBM3-SUMO-Defensins expression in*E. coli*SHuffle T7. The molecular weight of the CBM3-SUMO-Defensins is 33.8kDa，34.0 kDa, 33.9kDa，35.5kDa, as indicated with arrows.

Verification of CBM3-SUMO-Defensins Function

4 types of CBM3-SUMO-Defensins were subjected to salt removal by gradient dialysis, followed by cleavage with recombinant Ulp1 (Beyotime, P2312S). The results from SDS-PAGE electrophoresis showed a slight decrease in the molecular weight of the target protein (Fig. 5 A), indicating successful removal of ~4 kDa defensin peptides. Since CBM3-SUMO-Defensins would ultimately be incorporated into wound dressing products in a domain-bound form rather than as individual defensins, we did not further purify the defensins. Instead, we utilized the enzyme-cleaved CBM3-SUMO-Defensins (designated as CBM3-SUMO↓Defensins) for antimicrobial assays. As depicted in Fig. 5b, Escherichia coli and Staphylococcus aureus were selected as representatives of Gram-positive and Gram-negative bacteria, respectively. The CBM3-SUMO↓Defensins cleaved by Ulp1 enzyme exhibited antimicrobial activity against both strains, while the uncleaved CBM3-SUMO-Defensins showed no antimicrobial activity. This suggests that we successfully produced active defensin molecules using the fusion protein cleavage approach.

![](https://static.igem.wiki/teams/5185/figures/eng/engineering-fig-5.png)

Figure 5 (a) Verification of SUMO cleavage of CBM3-SUMO-Defensins. -Ulp1 represents CBM3-SUMO-Defensins that have not been treated with Ulp1, +Ulp1 represents CBM3-SUMO↓Defensins cleaved by Ulp1. (b) Antimicrobial assays of four types of CBM3-SUMO↓Defensins against Escherichia coli and Staphylococcus aureus

Subsequently, during our interview with Dr. Dai, we learned that the market approval of antimicrobial drugs requires the determination of Minimum Inhibitory Concentration (MIC). Therefore, we utilized the microdilution method to determine the MIC values of four types of CBM3-SUMO-Defensins. For specific details, please refer to our measurement section. Initially, we examined the 24-hour growth curves of Staphylococcus aureus with the addition of CBM3-SUMO↓Defensins. Within the 0–8 hour range, all four types of CBM3-SUMO↓Defensins exhibited antimicrobial activity (Fig. 6A). We selected the 8-hour time point to define the MIC values against Staphylococcus aureus. At this point, the MIC50 values for CBM3-SUMO↓HNP1, CBM3-SUMO↓HNP4, CBM3-SUMO↓HD5, and CBM3-SUMO↓HBD3 were 0.74 μM, 0.368 μM, 1.475 μM, and 1.001 μM, respectively. Additionally, the MIC90 values for CBM3-SUMO↓HNP4/HD5 were 0.735 μM and 1.475 μM, respectively. These values are close to the MIC values reported in the literature for the four defensins (Wei et al., 2009).

![](https://static.igem.wiki/teams/5185/figures/eng/engineering-fig-6.png)

Figure 6: Verification of the antibacterial activity of CBM3-SUMO↓Defensins. (a) The effect of the four CBM3-SUMO↓Defensins on the growth of Staphylococcus aureus within 24 hours. (b) The inhibition rate of CBM3-SUMO↓Defensins on Staphylococcus aureus after 8 hours.

It is worth mentioning that when the concentrations of the four types of CBM3-SUMO↓Defensins were reduced to 185 nM, 184 nM, 184 nM, and 125 nM, they were able to promote the growth of Staphylococcus aureus (Fig. 6 B). This suggests that the non-defensin portion of CBM3-SUMO↓Defensins may serve as a nutrient for bacteria, providing amino acids upon hydrolysis. After 8 hours, high concentrations of CBM3-SUMO↓Defensins were able to promote the growth of Staphylococcus aureus (Fig. 6 A). We speculate that this is due to the short peptide nature of defensins, which makes them susceptible to degradation by proteases, resulting in a shorter effective period. After defensins become inactive after 8 hours, CBM3-SUMO↓Defensins act as nutrients that promote bacterial growth. Therefore, in our antibacterial dressings, a higher concentration is not necessarily better. We believe that in the future, we can choose smaller Binding domains or optimize the sequence of natural Binding domains to increase the proportion of defensin molecules as much as possible while keeping the molar concentration of the fusion protein constant, thereby reducing the non-defensin portion to avoid providing nutrients to bacteria and improving the MIC. When we consulted Dr. Ren, he informed us that all antibacterial dressings need to be changed within 12 hours. This is not a defect, but it should be stated in the product instructions.

DBTL Cycle IV: Universality of Antimicrobial Binding Domains

After separately validating the effectiveness of the binding domain and defensins, we expanded CBM3-SUMO↓Defensins to other binding domains by replacing CBM3 with the functionally validated α2, CBM5, and CBMxx. This allows for binding to collagen, chitosan, and alginate, thereby creating a more versatile collection of antibacterial dressings, which enhances the potential of our first aid kit to address more complex situations.

Construction of binding domain-SUMO-HNP1 strain

We assembled the binding domain fragments from DBTL Cycle I with the SUMO and HNP1 fragments from DBTL Cycle 2 using Golden Gate assembly, to obtain SHuffle T7 strains that express α2-SUMO-HNP1, CBM2-SUMO-HNP1, CBM5-SUMO-HNP1, and CBMxx-SUMO-HNP1 (Fig. 7a). We then used the same fermentation method as for CBM3-SUMO-HNP1 to produce the corresponding fusion proteins (Fig. 7b). Among these, CBM5-SUMO-HNP1 and α2-SUMO-HNP1 were successfully expressed in the supernatant, but the yield was relatively low. In contrast, CBMxx-SUMO-HNP1 did not express, which we speculate may be due to contamination. Due to time constraints, we only tested the successfully expressed CBM5-SUMO-HNP1 and α2-SUMO-HNP1.

![](https://static.igem.wiki/teams/5185/figures/eng/engineering-fig-7.png)

Figure 7 (a) Identification of Binding domain-SUMO-HNP1 expressing strains: The lanes from left to right represent the SHuffle T7 expression strains for α1-SUMO-HNP1, α2-SUMO-HNP1, CBM2-SUMO-HNP1, CBM3-SUMO-HNP1, CBM5-SUMO-HNP1, CBM13-SUMO-HNP1, CBM16-SUMO-HNP1, CBM96-SUMO-HNP1, and CBMxx-SUMO-HNP1. (b) SDS-PAGE analysis of the recombinant defensins expression in*E. coli*SHuffle T7. The lanes from left to right are Marker, WT whole cell, CBM5-SUMO-HNP1 (20.3 kDa) cell lysate supernatant, CBM5-SUMO-HNP1 inclusion body, α2-SUMO-HNP1 cell lysate supernatant (38.0 kDa), α2-SUMO-HNP1 inclusion body, CBMxx-SUMO-HNP1 (39.1 kDa) cell lysate supernatant, and CBMxx-SUMO-HNP1 inclusion body.

Verification of binding domain-SUMO-HNP1

We used the same cell lysis purification method as CBM3-SUMO-HNP1 to purify CBM5-SUMO-HNP1 and α2-SUMO-HNP1. Due to their poor solubility, we increased the amount of the upper column by two-fold. Subsequently, we performed SUMO cleavage and antibacterial tests using the same procedure. Both CBM5-SUMO↓HNP1 and α2-SUMO↓HNP1 demonstrated inhibitory effects on Escherichia coli and Staphylococcus aureus. Therefore, we believe that the designed Binding domain-SUMO-Defensins are scalable.

![](https://static.igem.wiki/teams/5185/figures/eng/engineering-fig-8.png)

Figure 8 Antibacterial assay of α2-SUMO↓HNP1 and CBM5-SUMP↓HNP1 against*E. coli*and *S. aureus*

DBTL Cycle V: Application

After validation by DBTL III and IV, the feasibility of our Binding domain-SUMO-Defensins in antimicrobial activity has been demonstrated. Next, we need to move it into practical application by attaching the defensin to wound dressings through the binding domain and releasing the defensin for antimicrobial activity through SUMO cleavage.

However, we will face a practical challenge, that is how to use the SUMO enzyme? In preliminary experiments, we used commercially purchased recombinant yeast Ulp1, where 1 uL (10 U) can be used to cleave 20 ug of fusion protein. Based on our MIC determination results, it is estimated that 1-2 enzyme cleavage reactions are needed to produce one piece of antimicrobial dressing, while the cost of the SUMO enzyme is approximately 10-20 RMB. The cost of such antimicrobial dressings is too high that no consumer will choose them, especially in war zones and disaster areas that require humanitarian aid.

Expression and enzyme digestion verification of rtUlp1

To reduce costs, we decided to produce recombinant Ulp1 from Escherichia coli to lower costs. Based on this, we constructed a truncated Ulp1 strain (rtUlp1) that retains the enzyme's active center (Wang et al. 2016) and achieved soluble expression in a 400 mL flask (Fig.9A).

![](https://static.igem.wiki/teams/5185/figures/eng/engineering-fig-9.png)

Figure 9 Expression and validation of rtUlp1. (a)SDS-PAGE identification of soluble expression of rtUlp1，where S represents the supernatant of cell lysate and IB represents inclusion bodies. (b) Identification of enzymatic activity of rtUlp1.

Subsequently, we performed the enzyme digestion reaction under the same conditions as the purchased Ulp1 enzyme. This experiment was carried out simultaneously with the optimization experiment of HNP1 (link to modeling), so we chose CBM3-SUMO-HNP1AWK as the substrate and conducted antibacterial experiments on E. coli. The results are shown in Fig. 9B. After our expressed rtUlp1 processed CBM3-SUMO-HNP1AWK, it displayed the same inhibition zone as the CBM3-SUMO↓HNP1AWK processed by Ulp1, indicating that our expressed rtUlp1 has SUMO enzyme cutting activity.

In this experiment, we took 200 mL of the fermentation broth from the 400 mL shaker flask for purification, and obtained 3 mL of rtUlp1 (concentration not tested). We then added 1 μL to successfully achieve the enzyme-mediated release of the defensin. Therefore, we believe that the cost of producing our own Ulp1 is extremely low, and it can be mass-produced for wound dressings. To extend the shelf life of rtUlp1, we decided to store it in powder form with water, together with the wound dressing that connects the Binding domain-SUMO-Defensins, as described in the [description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D).

Binding domain-rtUlp1 design

![](https://static.igem.wiki/teams/5185/figures/eng/engineering-fig-10.png)

After showcasing the above results and the product to Mr. Dawa again, he was highly impressed and provided valuable feedback: the current usage is too cumbersome, as the injured person or medical staff would need to carefully mix the water with the rtUlp1 powder, and then combine the rtUlp1 solution with the wound dressing. This would be difficult for injured hands or in urgent situations, making it less applicable than traditional dressings and antibiotics. Therefore, we decided to optimize the design.

![](https://static.igem.wiki/teams/5185/figures/eng/engineering-fig-11.png)

Our solution is to adopt the same immobilization strategy as the defensins, by fusing rtUlp1 with the Binding domain. The rtUlp1 can be placed at either the N-terminus or C-terminus of the fusion protein, and then combined with the Binding domain-SUMO-Defensins on the wound dressing through freeze-drying. By immobilizing the rtUlp1 via the binding domain, the distance between rtUlp1 and the SUMO tag will be reduced, which may enhance the enzyme cutting activity of rtUlp1, further reducing the production cost.

References

Wang, X., Liu, H., Liu, Y., Li, Y., Yan, L., Yuan, X., ... & Chu, Y. (2016). A novel strategy for the preparation of codon-optimized truncated Ulp1 and its simplified application to cleavage the SUMO fusion protein. The Protein Journal, 35, 115-123.
Wei, G., de Leeuw, E., Pazgier, M., Yuan, W., Zou, G., Wang, J., ... & Lu, W. (2009). Through the looking glass, mechanistic insights from enantiomeric human defensins. Journal of Biological Chemistry, 284(42), 29180-29192.
Xie, Q., Wang, Y., Zhang, M., Wu, S., Wei, W., Xiao, W., ... & Xu, P. (2022). Recombinant HNP-1 produced by Escherichia coli triggers bacterial apoptosis and exhibits antibacterial activity against drug-resistant bacteria. Microbiology Spectrum, 10(1), e00860-21.
Zhu, S., Weber, D. K., Separovic, F., & Sani, M. A. (2021). Expression and purification of the native C‐amidated antimicrobial peptide maculatin 1.1. Journal of Peptide Science, 27(8), e3330.

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Molecular Experiments](#d1)
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Fermentation Experiment](#d2)
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Purification Experiment](#d3)
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Zone of Inhibition Experiment](#d4)
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Hydrogel & Cellulose Binding Experiments](#d5)
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Yeast Expression Experiment](#d6)
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Broth Diluton Experiment](#d7)

Molecular Experiment

1. PCR

- Annealing temperature: primer Tm value -5
- Enzyme: PfuDNA polymerase
- Extension time：15s/kb

2. DNA Gel purification

- Measure DNA concentration after purification

3. Goldengate assembly

- Using restriction enzymes BsaI and T4 DNA ligase
- Vector-segment ratio of 1:3

4. Transformation

- All goldengate products are transformed
- DH5α strain for plasmid cloning and
- BL21 for protein expression and synthesis

5. Colony PCR

- Pick single colony as template for colony PCR
- Annealing temperature: primer Tm value -5
- Enzyme: TaqDNA polymerase
- Extension time：15sec/kb

6. Sanger DNA sequencing

- Send positive bacterial strains tested by colony PCR to Rui Biotech Ltd. Guangzhou for DNA sequencing for confirmation of correct sequence

7. Strain preservation

- Preserve strain by mixing bacterial liquid with glycerol in 1:1 ratio (e.g. 100ul bacterial liquid mix with 100ul glycerol)

8. Plasmid extraction

- Alkaline lysis method

List of Primers

List of Plasmids & Strains

Fermentation Experiment

1. Activation

- Pick a single colony from the plate and inoculate it into a 4 mL LB medium containing K-antibiotic in a shaking tube.
- Prepare 400 mL LB medium in a 1 L shaking flask (No need for a pET-28a control)

2. Scale-up Culture

- Add 200 µL of K-antibiotic to the 400 mL shaking flask, then add the entire 4 mL culture from Day 3. Incubate at 37°C with shaking at 220 rpm for 2 hours.

3. Induction

- When the OD600 of the culture in the shaking flask reaches 0.6-0.7 (the liquid becomes semi-opaque, such that your fingers appear slightly blurred when viewed through it), add 400 µL of 1M IPTG and incubate at 37°C for 3.5-4 hours

4. Harvesting Cells

- Collect the culture in a 200 mL centrifuge bottle and centrifuge at 8500 rpm for 10-15 minutes. Discard the supernatant and add 40 mL of 20 mM Tris-HCl

5. Cell Disruption

- Disrupt the cells at 80% amplitude for 25 minutes

6. Centrifugation

- Centrifuge at 8500 rpm for 10 minutes at 4°C to separate the inclusion bodies (pellet) and the cell lysate (supernatant)

Purification Experiment

1. Reagent preparation

- Washing solution: 20mM Tris HCl+20mM imidazole
- Eluent: 20mM Tris HCl+200mM imidazole
- 20% ethanol

2. Material preparation

- Fill with new column material, 3mL denaturation-resistant column material.
- The column material can be reused and stored in 20% ethanol in a 4℃ refrigerator
- Open the stopper and let the ethanol in the column material flow out naturally
- Add 20mM Tris-HCl, fill it up, let it flow out naturally, repeat 3 times, and wash the residual ethanol in the column material
- Plug the stopper for use

3. Purification

- Add the broken cell lysate (supernatant) to the column material, collect the effluent liquid in a 50 mL centrifuge tube, and the collected liquid is the flow-through liquid (FT). Observe whether the FT becomes clear. If not, add it to the column material again until the flow-through liquid becomes clear. The more flow-through times, the better
- Fill the column material with cleaning solution, open the stopper, add new cleaning solution after it flows out, repeat twice, and mark the collected cleaning solution as W
- Add 2~3 times the volume of elution solution to the column material (3 mL column material plus 8 mL elution solution), open the stopper, discard the 1 mL of liquid that flows out first, and then start collecting the effluent, marked as E

4. Post-treatment of column material

- Wash with elution solution for another 2-3 times the column volume to remove residual protein
- Wash with 20 mM Tris-HCl for 4-5 times the column volume
- After washing with 20% ethanol 2-3 times the column volume, fill it with 20% ethanol and store it in refrigerator at 4

5. Ultrafiltration\* (not recommended, protein will precipitate)

- Add the eluted solution in the 15 mL centrifuge tube to the upper layer of the ultrafiltration tube and fill it up to the mark with Tris-HCl
- Place the ultrafiltration tube in a precooled centrifuge below 10°C and centrifuge at 6500 rpm for 10 min
- Transfer the filtrate from the lower layer of the ultrafiltration tube to a new 50 mL centrifuge tube, mark it as filtrate, and collect 1 mL into a clean 1.5 mL centrifuge tube
- Fill the upper layer of the ultrafiltration tube with Tris-HCl to the mark. (If there is little filtered liquid, use a micropipette to blow the ultrafiltration membrane to resuspend the insoluble particles)
- Repeat b-d until the filtrate volume reaches 50 mL
- Transfer the upper layer of the ultrafiltration tube to a new 15 mL centrifuge tube, and be careful to transfer the insoluble particles on the filter membrane together, and mark it as concentrated

6. Run the flow-through, washing, elution, concentration, and filtrate through a protein gel

7. Dialysis (we recommend gradient dialysis)

- Prepare dialysate
#1: final concentration of 100 mM imidazole dissolved in 20 mM TrisHCl Dialysate
#2: final concentration of 50 mM imidazole dissolved in 20 mM TrisHCl Dialysate
#3: final concentration of 25 mM imidazole dissolved in 20 mM TrisHCl
- Soak the dialysis bag in pure water and boil for 1 minute, pour out the boiling water, add pure water, and check whether the dialysis bag is leaking
- Pour out the pure water, add dialysate #1, wait for the dialysis bag to cool down, carefully pour the eluted protein into the dialysis bag, clamp the clamp, and check for leakage
- Soak the dialysis bag completely in the dialysis solution and place it in a 4℃ refrigerator for 2h
- Pour out the dialysis solution #1, add the dialysis solution #2, and place it in a 4℃ refrigerator for 2h
- Pour out the dialysis solution #2, add the dialysis solution #3, and place it in a 4℃ refrigerator for 2h
- Pour out the dialysis solution #3, add pure water, and place it in a 4℃ refrigerator for 2h

8. SUMO digestion

- Use the BCA protein assay kit to determine the protein concentration
- Configure the SUMO digestion system on ice
  - Sumo-tag protein, 20 ug
  - 10×sumoBuffer (+Salt/-Salt), 20 uL
  - Ulp1 protease (+Salt/-Salt), 0.5 uL
  - ddH20, up to 200 uL
- 4℃ overnight reaction
- Protein electrophoresis verification

Zone of Inhibition Experiment

- Inoculate Staphylococcus aureus and Escherichia coli into 5 mL of antibiotic-free LB, 220 rpm, 37°C overnight
- Take 200 uL of bacterial solution and evenly spread it on the antibiotic-free LB plate
- After the bacterial solution on the plate evaporates, poke a hole with a 1 mL sterile pipette tip
- Drop the antimicrobial drug to be tested into the small hole and let it evaporate
- Invert the plate in a 37°C incubator overnight

Hydrogel & Cellulose Binding Experiments

Collagen/chitosan binding

- Configure 4.5% collagen/chitosan water solution, stir and dissolve at 70℃ ~30min
- Add 4.5% sodium alginate to the prepared collagen solution, stir and dissolve at 90℃ for ~3h until completely dissolved, and leave it overnight for swelling
- Add the binding domain-fluorescent fusion protein to the collagen mixture, stir and dissolve at room temperature ~ 10min, until the colour is uniformly displayed and no dominant sediment presented. Vacuum dry the solution ~1h
- Evenly distribute the mixture on the median dish, add 0.4% calcium chloride solution for solidification, the target hydrogel will be obtained overnight
- Observe and compare protein binding efficiency under blue-light

Cellulose binding

- Add 2 rows of protein (different binding domain proteins linked to chromoprotein and chromoprotein as control) to 48-well plate
  - Row 1: gauze soaked in protein
  - Row 2: protein only
- Soak overnight at 4°C
- Transfer gauze in Row 1 to isopropanol
- Dry at 40°C
- Wash with water
- Observe and compare protein binding efficiency under blue-light

Yeast Expression Experiment

Pichia pastoris competent cell preparation

1. Inoculate a single colony of yeast recipient strain on YPD plate and culture at 30°C for 2 days;
2. Pick up the single colony from the plate and inoculate it into 5mL YPD liquid medium, shake at 30°C overnight;
3. After overnight culture, inoculate at an initial OD600 of 0.2 into 50mL YPD medium and shake culture to an OD value of 1.2~1.5 (about 5h+);
4. Centrifuge at 4°C, 5000rpm for 5 minutes to collect the sedimented cells, and resuspend the cells in 50ml of pre-cooled sterile water;
5. Centrifuge at 4°C, 5000rpm for 5 minutes to collect the sedimented cells, and resuspend the cells in 50ml of pre-cooled sterile water;
6. Centrifuge again at 4°C, 5000rpm for 5 minutes to collect the sedimented cells, and resuspend the cells in 10ml of 1mol/L sorbitol;
7. Solubilize the cells in 500ul of 1mol/L pre-cooled sorbitol, do not add glycerol, and store at -80°C for a few hours until transformation.

Pichia pastoris electroporation

1. Prepare 80 µL of yeast competent cells and mix them with 5-10 µg of purified linearized plasmid on ice for 15 minutes (ice bath for pre-cooling the electroporation cup).
2. Electroporate the mixture (1500 V, 400 Ω, 25 F).
3. After electroporation, quickly add 1 mL of sorbitol (pre-cooled) and culture on a shaker at 30°C for 2 hours. Then, plate the cells on MD plates.
4. Grow the cells on MD medium at 30°C for 3-4 days and perform identification.

Selection for multi-copy transformants following electroporation

1. A spreader was used to suspend the transformants in sterile water.
2. After measuring cell density, cells were diluted accordingly so that the cells were plated at a density of 10 cells per standard 8.5 cm petri dish on YPD agar containing G418 (0 to 2 mg/ml).
3. Since the number of resistant colonies drops significantly above 0.5 to 1 mg/ml G418, a range of concentrations of G418 (e.g., 0.25, 0.5, 1 and 2 mg/ml) was used. It took two to five days for resistant transformants to appear.

Analyze samples by SDS-polyacrylamide gel electrophoresis

1. Thaw cell pellets quickly and place on ice.
2. For each 1 mL sample, add 100 uL Breaking Buffer to the cell pellet and resuspend.
3. Add an equal volume of acid-washed glass beads (size 0.5 mm). Estimate equal volume by displacement.
4. Vortex 30 seconds, then incubate on ice for 30 seconds. Repeat for a total of 8 cycles.
5. Centrifuge at maximum speed for 10 minutes at 4°C. Transfer the clear supernatant to a fresh microcentrifuge tube.
6. Take 50 uL of supernatant (cell lysate) and mix with an appropriate volume of denaturing PAGE Gel Loading buffer (Sample Buffer).
7. Heat the sample as recommended and load 10-20 uL per well. Thickness of the gel and number of wells will determine volume loaded. You may store the remaining sample at -20°C for western blots, if necessary. You may store the cell lysates at -80°C for further analysis.

Yeast Extract Peptone Dextrose Medium (1 liter)

1. Dissolve 10 g yeast extract and 20 g of peptone in 900 mL of water. Note: Add 20 g of agar if making YPD slants or plates.
2. Autoclave for 20 minutes on liquid cycle.
3. Add 100 mL of 10X D.
4. Store the liquid medium at room temperature. Store the YPD slants or plates at 4°C. The shelf life is several months.

Broth Diluton Experiment

1. Preparation of Inoculum:

- Grow the microbial strain overnight in appropriate broth at the optimal temperature.
- Dilute the overnight culture to achieve a final concentration of approximately 1 x 10^6 CFU/mL (colony-forming units per milliliter).

2. Preparation of Antimicrobial Dilutions:

- Prepare a stock solution of the antimicrobial substance at a concentration that is 10 times higher than the expected MIC.
- Perform serial dilutions of the stock solution in the appropriate broth to achieve a range of concentrations (e.g., 0.5, 1, 2, 4, 8, 16, 32, 64 µg/mL).

3. Setting Up the Microtiter Plate:

- Dispense 100 µL of the diluted antimicrobial solutions into the wells of a 96-well microtiter plate.
- Include wells with only broth as negative controls and wells with the microbial inoculum and no antimicrobial as positive controls.

4. Inoculation:

- Add 100 µL of the standardized microbial inoculum (1 x 10^6 CFU/mL) to each well containing the antimicrobial dilutions.
- Mix gently by pipetting up and down or using a vortex mixer.

5. Incubation:

- Incubate the microtiter plate at the appropriate temperature for the specific microorganism (usually 24 hours for bacteria at 35-37°C or 48 hours for fungi at 25-30°C).

6. Reading the Results:

- After incubation, check each well for microbial growth, which can be indicated by turbidity or color change (if using indicator dyes).
- The MIC is defined as the lowest concentration of the antimicrobial agent that prevents visible growth in the well.

7. Data Analysis:

- Record the MIC for each antimicrobial tested.
- Repeat the experiment at least twice to confirm results and calculate the average MIC.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](https://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project and what you learned from them. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort. Furthermore, you can still impress the judges by thoughtfully discussing what went wrong and how you might change things.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}