iGEM 2025 — Team cau-china

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{% block title %}Notebook{% endblock %}
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{% block nav\_text\_1 %}▶ Notebook1{% endblock %}
{% block nav\_text\_2 %}▶ Notebook2{% endblock %}
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# Notebook1

[Donwload PDF](https://static.igem.wiki/teams/5846/wiki/notebook/notebook-1.pdf)。

# Notebook2

[Donwload PDF](https://static.igem.wiki/teams/5846/wiki/notebook/notebook-2.pdf)。

# Notebook3

[Donwload PDF](https://static.igem.wiki/teams/5846/wiki/notebook/notebook-3.pdf)。

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{% block title %}Engineering{% endblock %}
{% block lead %}https://static.igem.wiki/teams/5846/wiki/header-image/engineering.webp{% endblock %}
{% block nav\_text\_1 %}▶ Overview{% endblock %}
{% block nav\_text\_2 %}▶ Wet Lab{% endblock %}
{% block nav\_text\_2\_1 %}◆ Overview{% endblock %}
{% block nav\_text\_2\_2 %}◆ Verification of the Synthesis and Infectivity of Phage-like particles{% endblock %}
{% block nav\_text\_2\_3 %}◆ Reconstructing the Phage Genome{% endblock %}
{% block nav\_text\_2\_4 %}◆ Tail fiber replacement: Phage infection{% endblock %}
{% block nav\_text\_3 %}▶ Dry Lab{% endblock %}
{% block nav\_text\_3\_1 %}◆ Overview{% endblock %}
{% block nav\_text\_3\_2 %}◆ Software{% endblock %}
{% block nav\_text\_4 %}▶ Human Practice{% endblock %}
{% block nav\_text\_4\_1 %}◆ Overview{% endblock %}
{% block nav\_text\_4\_2 %}◆ SynBio Halli Galli Engineering{% endblock %}
{% block nav\_text\_4\_3 %}◆ CHUN GENG Program Engineering{% endblock %}
{% block nav\_text\_5 %}▶ References{% endblock %}
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# Overview

Our project rigorously adheres to the **“Design-Build-Test-Learn”** (DBTL) engineering cycle, a framework that guides all aspects of our work, from wet and dry lab research to integrated Human Practices.

![Figure 0](https://static.igem.wiki/teams/5846/wiki/engineering/fig0.webp)

In the ***Design*** phase, we design not only biological systems but also our public engagement and education strategies, while identifying **key knowns and unknowns in both technical and societal contexts**.

During the ***Build*** phase, we assemble biological parts in the lab while also constructing platforms for dialogue with communities, experts, and stakeholders.

The ***Test*** phase involves validating the function of biological components and gauging public understanding and acceptance of our technology through interactive activities.

Finally, in the ***Learn*** phase, we synthesize insights from both experimental data and societal feedback to close the loop.

This comprehensive and iterative engineering approach ensures our project advances responsibly on both scientific and social fronts, embodying the **“middle-out”** philosophy essential to synthetic biology.

# Wet Lab

## Overview

We follow a step-by-step development process for each section, progressively constructing a programmable phage-like particle platform. We designed and validated the synthesis and DNA delivery capabilities of phage-like particles; Building upon this foundation, we engineered a stable and controllable chassis organism using genome integration techniques. Finally, through modular tail-reprogramming, we achieved specific targeting of novel hosts by the phage-like particles. Layered validation confirmed their specific recognition and infection of target pathogens. Each iteration deepened our understanding of the system’ s controllability and functional programmability, driving continuous refinement of the platform.

## **Verification of the Synthesis and Infectivity of Phage-like particles**

To address the uncontrollable self-replication of natural phages, we plan to replace the natural genome within the head coat of the wild-type T7 phage with DNA concatemer formed by rolling-circle replication of the virulent plasmid. The resulting phage-like particles retain the ability to infect target bacteria and deliver toxin genes, leading to host cell death. Since the DNA fragment lacks genes for phage coat synthesis, these particles cannot produce progeny, thereby enabling controlled bactericidal activity. To visualize the synthesis and infectivity of the phage-like particles during validation, we replaced the toxin gene *MazF* on the plasmid with *bjGFP* (a gene encoding green fluorescent protein), and conducted the following experiments.

![Fig. 1](https://static.igem.wiki/teams/5846/wiki/engineering/f1.avif)

**Figure 1: The model of packaging visualization plasmid**

### **1st Iteration**

#### **Design**

We aim to infect *E. coli* MG1655 containing pUC19-packaging-PTetA-bjGFP with WT T7 to obtain T7 Mix containing both WT T7 and phage-like particles. Subsequently, we infected WT *E. coli* MG1655 (untransformed with the plasmid) using the T7 Mix. Ultimately, we observed bjGFP expression in the bacteria, with colonies emitting green fluorescence.

#### **Build**

We cultured *E. coli* MG1655 containing the pUC19-packaging-PTetA-bjGFP plasmid to an OD600 of 0.7, added 4×10¹⁰ PFU/mL of the WT T7, and incubated at 37 °C with shaking until most bacteria lysed. Add 0.2 mL chloroform, vortex vigorously, centrifuge, and collect the supernatant. Filter through a 0.22 μm membrane to obtain the T7 Mix.

Subsequently, we cultured uninoculated WT *E. coli* MG1655 to an OD600 of 0.5 and placed it on ice. We performed a 10⁻¹³ serial dilution of the T7 Mix, with the final well containing LB as a blank control. Take 20 μl of each dilution and mix with 20 μl of recipient bacterial suspension or LB (cell-free control). Incubate at 37 °C with shaking for 1 hour. Plate 10 μl of each mixture onto the LB plates containing 50 μg/mL Amp and incubate at 37 °C overnight.

#### **Test**

In all the dilution gradient experimental groups, no colonies were observed growing on LB plates containing Amp. Simultaneously, no colony growth was observed in the cell-free control group, ruling out the possibility of medium contamination.

#### **Learn**

Upon careful literature review, we discovered that the successful construction of similar packaging systems referenced in our work utilized the BW25113 Δ*trxA* strain. The thioredoxin encoded by the *trxA* gene serves as a host factor essential for the functional activity of the T7 phage DNA polymerase. In the Δ*trxA* strains, T7 DNA polymerase cannot be effectively “activated,” resulting in extremely low replication efficiency. Consequently, wild-type T7 phage cannot complete effective genomic replication in this host, with its life cycle blocked at the DNA replication stage, significantly inhibiting its ability to lyse bacteria.

In contrast, the WT *E. coli* MG1655 strain used in our experiments possesses an intact *trxA* gene. Consequently, when T7 Mix infects WT *E. coli* MG1655, the numerically dominant wild-type T7 phages with full replication capacity rapidly complete the entire cycle of adsorption, injection, replication, assembly, and lysis.

We hypothesize that the rapid and efficient lysis by WT T7 phage eliminates the majority of host cells before they can stably take up and express the DNA injected by phage-like particles. This results in the absence of any surviving colonies on plates containing ampicillin.

### **2nd Iteration**

#### **Design**

To minimize interference from the WT T7 lysing WT *E. coli* MG1655 during the final assessment of phage-like particle infectivity, we attempted to modify the final detection method. After obtaining the T7 Mix, we mixed WT *E. coli* MG1655 with the T7 Mix to create the double-layer plate, hoping to observe colonies emitting green fluorescence on the upper layer.

#### **Build**

We obtained the T7 Mix using the same method as the previous iteration. Then, 100 μl of OD600=0.7 WT *E. coli* MG1655 was mixed with 100 μl of the T7 Mix. After incubating at room temperature for 10 min, we added 4 ml of LB containing 0.7% agar and 50 μg/ml Amp, poured it onto a 1.5% agar plate, and cultured it at 37 °C for 12 hours to observe the results.

#### **Test**

In the WT *E. coli* MG1655 + T7 Mix group, we observed colony growth but no fluorescence. The control group (LB + T7 Mix) showed no colony growth, ruling out the possibility of contaminating bacteria. We picked colonies from the G1 plate and cultured them overnight in liquid LB containing 50 μg/mL Amp, hoping to observe the phenotypic expression of bjGFP. Unfortunately, after overnight incubation, the liquid remained completely clear with no bacterial growth.

![Fig. 2](https://static.igem.wiki/teams/5846/wiki/engineering/fig-2.webp)

**Figure 2: Left (G1): WT *E. coli* MG1655+T7 Mix, Right (G5): LB+T7 Mix**

#### **Learn**

Based on the above results, we speculate that the bacterial growth in liquid medium is inhibited because WT T7 inevitably contaminates the plate during colony picking. WT T7 proliferates extensively and lyses the host, preventing the host bacteria from growing in the liquid medium. Therefore, we are unable to verify the synthesis and infectivity of phage-like particles using the double-layer plate method.

### **3rd Iteration**

#### **Design**

To more convincingly validate the synthesis and infectivity of the bacteriophage-like particles, we attempted a novel detection method. After obtaining the T7 Mix, we co-cultured the WT *E. coli* MG1655 with the T7 Mix in the liquid medium for a period of time, expecting the bacteriophage-like particles to inject the DNA within their capsids into the WT *E. coli* MG1655 during this period. Subsequently, we resuspended the bacteria in LB medium, washing away as much T7 Mix as possible. The cells were then spread onto LB plates containing 50 μg/mL Amp and incubated at 37 °C overnight. We anticipated observing colonies that would grow and emit green fluorescence.

#### **Build**

We prepared the T7 Mix using the same method as the previous iteration. Then, we mixed OD600=0.6 WT *E. coli* MG1655 with the T7 Mix at the three different ratios, cultured the mixture in liquid LB for 1.5 hours, collected the cells, and resuspended them three times in LB to wash away as much T7 Mix as possible. Finally, the cells were spread onto LB plates containing 50 μg/ml Amp and incubated overnight at 37 °C.

#### **Test**

In all three experimental groups with different mixing ratios, no colony formation was observed. The plates remained clear, inconsistent with the expected growth of green fluorescent colonies.

#### **Learn**

Based on these results, we hypothesize that the absence of colonies on LB plates containing 50 μg/ml Amp is due to the high proportion of WT T7 in the T7 Mix. During co-culture with WT *E. coli* MG1655, the majority of WT *E. coli* MG1655 was infected by WT T7. We were unable to sufficiently reduce the interference of WT T7 in the validation infection experiment, thus failing to observe the desired phenotype.

### **4th Iteration**

#### **Design**

Due to the unfavorable progress of previous infection experiments, we adopted the stratified validation approach to verify whether the plasmid rolling circle replication-formed DNA concatemer was successfully packaged within the head capsid of the phage-like particles in the T7 Mix, thereby confirming the successful synthesis of the phage-like particles. Simultaneously, in experiments verifying the infectivity of the phage-like particles, we attempted to incorporate a phage inhibitor to mitigate the influence of wild-type T7 during co-culture with WT *E. coli* MG1655. This phage inhibitor demonstrates varying degrees of suppression against a broad spectrum of phages while allowing normal bacterial growth and protein expression.

#### **Build**

For verifying phage-like particle formation, we obtained the T7 Mix using the same method as previous iterations. A portion of T7 Mix underwent heat lysis to release DNA from the particle capsids. The M13-F and M13-R primers were then selected for PCR amplification to detect specific bands.

For verifying phage-like particle infectivity, we cultured WT *E. coli* MG1655 with phage inhibitor added at a 1:500 ratio. When OD600 reached 0.7, 8 ml of OD600=0.7 WT *E. coli* MG1655 was mixed with 300 μl T7 Mix. The mixture was cultured in liquid LB for 1.5 hours, then the cells were harvested and resuspended three times in LB to wash away as much T7 Mix as possible. Finally, the cells were spread onto LB plates containing 50 μg/ml Amp and incubated at 37 °C overnight.

#### **Test**

In the PCR reaction, our target specific band is 2028 bp. Lane 1 exhibited three bands, with the longest band matching the expected length. Sequencing results confirmed the target band’ s sequence is entirely correct.

![Fig. 3](https://static.igem.wiki/teams/5846/wiki/engineering/fig-3.webp)

**Figure 3: The results of PCR performed on T7 Mix after thermal cracking**

M: star marker D5000 ladder; 1: PCR products from the T7 Mix after thermal cracking

![Fig. 4](https://static.igem.wiki/teams/5846/wiki/engineering/fig-4.webp)

**Figure 4: The design of detection primer and the sequencing results**

After overnight incubation at 37°C, we observed the growth of target colonies emitting green fluorescence in all three replicate groups under the identical conditions across the three experimental groups, consistent with our expected results.

![Fig. 5](https://static.igem.wiki/teams/5846/wiki/engineering/fig-5.webp)

**Figure 5: Phage inhibitor-treated *E. coli* MG1655 co-cultured with T7 Mix**

#### **Learn**

On LB plates containing 50 μg/ml Amp, we obtained the expected results, confirming that DNA from the phage-like particles was successfully injected into WT *E. coli* MG1655 and that the reporter gene *bjGFP* was successfully expressed. However, in the electrophoresis gel, we observed two non-specific bands in addition to the target band, suggesting possible interference from impurity DNA in our T7 Mix. We analyzed the composition of the previously used T7 Mix and the entire heat-lysate process, realizing that the free target plasmid DNA might be present in the T7 Mix. This DNA cannot be removed using filter membranes or the heat-lysate process. Therefore, we need to further eliminate the interference to more robustly demonstrate the presence of target DNA within the phage-like particles.

### **5th Iteration**

#### **Design**

After obtaining the T7 Mix, we thoroughly treated it with the RNase and the DNase to digest any potential plasmid DNA and other impurities, yielding T7 Mix 2.0. Subsequently, we performed PCR reactions using the same parameters as the previous iteration to detect the specific bands, verifying the DNA within the phage-like particles. We then re-conducted experiments related to the infectivity of the phage-like particles using T7 Mix 2.0.

#### **Build**

After obtaining the T7 Mix, we added 20 μl RNase and 5 μl DNase, mixed thoroughly, and incubated at 37°C for 30 minutes. We then added the phage pellet, centrifuged, aspirated the supernatant, resuspended in LB, and collected the T7 Mix 2.0. Subsequently, we used T7 Mix 2.0 to perform the same experiments as before to validate the synthesis and infectivity of the phage-like particles.

#### **Test**

After ruling out interference from free plasmid DNA in the T7 Mix, we repeated the PCR reaction and obtained the results shown in Figure 6.

In the PCR reaction, we observed the target specific band, and sequencing results confirmed the target band’ s sequence was entirely correct. Simultaneously, PCR reactions performed on the control group (WT T7 lysate) did not yield any relevant specific bands.

![Fig. 6](https://static.igem.wiki/teams/5846/wiki/engineering/fig-6.webp)

**Figure 6: The results of PCR performed on T7 Mix 2.0 after thermal cracking**

M: star marker D5000 ladder; 1: The PCR products from thermally lysed T7 Mix 2.0; 2: The PCR products from thermally lysed WT T7 phage

After overnight incubation at 37 °C, the experimental group observed the growth of target colonies emitting green fluorescence, consistent with our expected results. The control group showed no colony growth, ruling out interference from extraneous microorganisms.

![Fig. 7](https://static.igem.wiki/teams/5846/wiki/engineering/fig-7.webp)

**Figure 7: Phage inhibitor-treated *E. coli* MG1655 co-cultured with T7 Mix 2.0**

#### **Learn**

After thorough RNase and DNase treatment of the T7 Mix, we eliminated interference from the free plasmid DNA and other contaminating DNA. Following heat-lysate PCR, we confirmed the successful synthesis of the phage-like particles. On the LB plates containing 50 μg/ml Amp, we observed target colonies emitting green fluorescence, confirming that the DNA within the phage-like particles was successfully injected into WT *E. coli* MG1655 and that the reporter gene *bjGFP* was successfully expressed.

## **Reconstructing the Phage Genome**

### **1st Iteration**

#### **Design**

The process of segmentally reassembling phage genomes onto *E. coli* involves three steps: first, obtaining phage genomic fragments; second, seamlessly cloning these fragments into the pKD46-SacB vector; and finally, performing double-exchange screening to integrate the genomic fragments into the *E. coli* genome.

For the first step - amplifying T7 phage genomic fragments - we propose treating the phage lysate supernatant with the thermal lysis method. This process denatures the phage capsid through thermal disruption, releasing the encapsulated DNA. This approach offers a straightforward and efficient method for obtaining phage genomic fragments. The treated liquid is directly used as a template to amplify T7g-1-1, T7g-1-2, T7g-2, T7g-3, T7g-4-1, and T7g-4-2. Homologous arms are added to the 5’ ends of the upstream and downstream primers to enable homologous recombination with the vector.

#### **Build**

After inoculating with phages, the bacterial lysate obtained from overnight cultures was filtered through a membrane to yield lysate supernatant. Following treatment at 100°C for 4 minutes, the lysate supernatant was directly used as template for PCR to amplify phage genomic fragments. We aimed to obtain PCR amplification templates through simple and rapid procedures and successfully acquire amplified genomic fragments.

#### **Test**

Gel electrophoresis analysis of our PCR products revealed that only T7g-1-1 was successfully amplified. Sanger sequencing of this fragment confirmed the accuracy of the product.

After confirming the partial feasibility of the thermal lysis approach, we repeated the experiment and attempted to incorporate a centrifugation step to precipitate proteins and other impurities. However, the results remained unsatisfactory: longer genomic fragments still failed to amplify successfully, and T7g-2 exhibited significant non-specific amplification.

![Fig. 8](https://static.igem.wiki/teams/5846/wiki/engineering/fig-8.webp)

**Figure 8: Results of PCR amplification of genomic fragments using the pyrolysis-treated supernatant as template**

M: Star Marker 1kb Ladder; 1: T7g-1-1; 2: T7g-1-2; 3: T7g-2; 4: T7g-3; 5: T7g-4-1

#### **Learn**

We suspect this may be due to excessive impurities in the pyrolysis-treated supernatant interfering with PCR, and possibly due to excessive thermal treatment causing fragmentation of the phage genome. Both factors could potentially lead to the PCR amplification failure.

### **2nd Iteration**

#### **Design**

To eliminate the influence of impurities in the lysate supernatant and obtain the complete T7 phage genome, we envisioned purifying the phage genome and then using it as a PCR template to attempt amplification of the phage genome fragment again.

#### **Build**

We extracted the T7 phage genome from the lysate supernatant using the phage genome extraction kit. After diluting it to 10 ng/μL, we used it as a PCR template to amplify genomic fragments.

#### **Test**

We performed the PCR amplification using the extracted phage genome as a template, amplifying the T7g-1-1, T7g-1-2, T7g-2, T7g-3, T7g-4-1, and T7g-4-2 genomic fragments. The PCR products were analyzed by electrophoresis and Sanger sequencing, both confirming correct results. All phage genomic fragments were successfully obtained.

![Fig. 9](https://static.igem.wiki/teams/5846/wiki/engineering/fig-9.webp)

**Figure 9: Agarose gel electrophoresis of individual genomic fragments amplified via phage genome PCR**

M1: Star Marker 1kb Ladder; 1: T7g-1-1; 2: T7g-1-2; 3: T7g-2; 4: T7g-2; 5: T7g-4-1; 6: T7g-4-2; M2: Star Marker D5000 Ladder

#### **Learn**

Our method for amplifying the extracted T7 phage genomic fragments proved viable, successfully incorporating the homologous arms required for seamless cloning at both ends of the fragments. The next step involves constructing the homologous recombination vector and verifying the proper expression of the gene fragments.

## **Tail fiber replacement: Phage infection**

### **Selection of replacement site**

### **1st Iteration****Design** Taking tomato bacterial spot disease as an example, to enhance the T7 phage’ s ability to specifically recognize and infect the pathogenic bacterium *Pseudomonas syringae*, we designed a strategy to replace the tail fibers of the T7 phage. We plan to employ homologous recombination technology to replace the key tail filament protein gene *gp17* in the T7 phage genome with the tail filament protein gene *HOQ98\_gp43* from the *Pseudomonas syringae* phage phi psa 17. This will completely replace the gp17 protein in its tail filament with the VO98\_215 protein, thereby improving its binding efficiency to the pathogen’ s outer membrane receptor. **Build** We selected 50 bp sequences upstream and downstream of the T7 phage *gp17* gene as the homologous arms for genomic homologous recombination. The *HOQ98\_gp43* gene was placed between these arms and inserted into the high-copy vector pUC19, constructing the recombinant vector pUC19-VO98\_215. Subsequently, wild-type T7 phage was used to infect *E. coli* MG1655 harboring the pUC19-VO98\_215 plasmid. Following infection, the T7 genome was injected into the host as linear double-stranded DNA and underwent homologous recombination with the fragment on the host’ s pUC19\_VO98\_215 plasmid. The *gp17* gene in its genome was replaced with the *HOQ98\_gp43* gene, releasing recombinant progeny with altered tails and tail filaments. Finally, we infected the target bacterium *Pseudomonas syringae* DC3000 with the obtained recombinant phage. ![Fig. 10](https://static.igem.wiki/teams/5846/wiki/engineering/f10.avif) **Figure 10: The model of Genome - Plasmid pUC19-VO98\_215 Homologous Recombination** **Test** Following the infection experiment, we observed no bacteriophages on the agar plates containing *Pseudomonas syringae* DC3000. Furthermore, the titer of the recombinant bacteriophage was lower than that of the wild-type T7 bacteriophage in parallel experiments. ![Fig. 11](https://static.igem.wiki/teams/5846/wiki/engineering/f11.avif) **Figure 11: Cycle1.1 Double-Layer Plating** **Learn** We conducted an in-depth analysis of the functional structure of the tail filament protein and reviewed extensive literature. Studies indicate that the N-terminal domain of the T7 bacteriophage’ s gp17 protein is responsible for connecting the tail filaments to the viral particle, while the C-terminal domain mediates binding to host receptors. Following the complete replacement of the gp17 protein in the tail filament with the VO98\_215 protein, the tail filament may fail to attach to the viral particle due to structural alterations. **2nd Iteration****Design** We identified that conventional protein sequence alignment methods were insufficient for determining substitution sites and strategies within the required timeframe, which led us to seek assistance from the dry lab team. They designed [Alphage](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27software%27%29%20%7D%7D), a software tool for the automated prediction of tail fiber protein substitution strategies. This software pinpointed the optimal insertion site for the heterologous tail substitution in the gp17 protein to be within the amino acid range of 140-150. Following this, we consulted the literature and utilized [Protein-Protein Docking Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) provided by the dry lab team to validate this series of sites. We ultimately selected the 149th residue as the final substitution site, based on its superior availability and post-substitution stability. The C-terminal region of the modified heterologous tail fiber specifically recognizes *Pseudomonas syringae*, while the N-terminal T7 fragment ensures stable attachment to the virus particle. ![Fig. 12](https://static.igem.wiki/teams/5846/wiki/engineering/fig-12.webp) **Figure 12: Sequence Alignment of Protein gp17 and VO98\_215 (ESPript 3.0)** **Build** We selected the 50-bp region upstream of the codon for amino acid 149 in the T7 phage *gp17* gene as the upstream homologous arm for genomic homologous recombination substitution, while the downstream homologous arm was identical to that used for full-length replacement. The segment of the *HOQ98\_gp43* gene spanning from the codon for amino acid 149 to the stop codon was positioned between the homologous arms and inserted into the high-copy vector pUC19, yielding the recombinant vector pUC19-VO98\_215(C-). Subsequent steps mirrored the full-length replacement protocol. Following T7 infection of the host bacterium, the downstream portion of the *gp17* gene in the genome was replaced with the *HOQ98\_gp43* gene, releasing genetically altered, heterozygous tail protein recombinant progeny. Finally, we infected the target bacterium *Pseudomonas dongfangensis* DC3000 with the obtained recombinant phage. ![Fig. 13](https://static.igem.wiki/teams/5846/wiki/engineering/f13.avif) **Figure 13: The model of Genome - Plasmid pUC19-VO98\_215(C-) Homologous Recombination** **Test** Following infection experiments, no plaques were observed on the *Pseudomonas syringae* DC3000 colony-containing plates. Moreover, the titer of the recombinant phage was lower than that of the wild-type T7 phage in parallel experiments. ![Fig. 14](https://static.igem.wiki/teams/5846/wiki/engineering/fig-14.webp) **Figure 14: Cycle1.2 Double-Layer Plating** **Learn** After careful analysis, we identified several potential causes: * Failure of homologous recombination * Inability to express the tail filament protein * Inability of the recombinant phage to adsorb, infect, or effectively lyse the target bacteria. All of these factors could potentially explain the absence of plaques observed on the *Pseudomonas syringae* DC3000 bacterial lawn plates. We recognize that more detailed experiments are required to systematically validate whether the tail replacement was successful and whether the resulting phage-like particles can effectively infect the target bacterium DC3000. **Tiered verification****Design** To address whether tail replacement was successful and whether the resulting phage-like particles could effectively infect the target bacterium DC3000, we designed experiments at the DNA and protein levels to validate tail replacement and phenotypic phage adsorption and infection of the target bacterium, respectively. **<DNA Level>** Specific primers were designed to validate, via PCR and sequencing, the presence of phages with successful genomic recombination and gene replacement in the recombinant progeny produced after T7 infection of host bacteria (*Escherichia coli* MG1655 (pUC19\_VO98\_215) and *Escherichia coli* MG1655 (pUC19\_VO98\_215(C-))). **<Protein Level>** Add a tag to the recombinant tail protein and use Western Blot to separate and verify the expression of the target protein. **<Adsorption Level>** Use transmission electron microscopy to capture the adsorption of recombinant phages onto DC3000. **<Infection Level>** Since phage plaques cannot be directly observed, other screening markers are used to indirectly verify the infection of DC3000 by recombinant phages. **Build** **<DNA Level>** Following T7 phage infection of host bacteria (*Escherichia coli* MG1655 (pUC19-VO98\_215) and *Escherichia coli* MG1655 (pUC19-VO98\_215(149-))), a liquid mixture containing recombinant progeny was produced. After filtration and sterilization, the phage genome was extracted. Specific primers rr1 and rr2 were designed for genomic PCR, with preliminary validation via electrophoresis. Subsequently, genomic PCR was performed using specific staggered primers VO98\_215/(150-)-up and VO98\_215/(150-)-down. After electrophoretic verification, sequencing and alignment were conducted. ![Fig. 15](https://static.igem.wiki/teams/5846/wiki/engineering/f15.avif) **Figure 15: Binding Status of Specific Primers** **<Protein Level>** In plasmids pUC19-VO98\_215 and pUC19-VO98\_215(C-), 6xCAT was inserted between the *HOQ98\_gp43* gene and downstream homologous arms, yielding two His-tagged plasmids: pUC19-VO98\_215(C-his) and pUC19-VO98\_215(C-)(C-his). A 6xHis tag was added to the successfully expressed tail filament protein. Following magnetic bead-based preliminary separation, the proteins were ultrafiltration-concentrated and finally separated and verified via Western Blot to assess target protein expression. ![Fig. 16](https://static.igem.wiki/teams/5846/wiki/engineering/f16.avif) **Figure 16: The model of His - Tag Plasmid** **<Adsorption Level>** Transmission electron microscopy (TEM) imaging was performed to visualize the adsorption of recombinant phage onto DC3000. A mixture of recombinant phage and DC3000 was incubated at a fixed ratio and used as the TEM sample. A control group featuring adsorption of wild-type T7 phage onto DC3000 at the same ratio was included to validate changes in the adsorption capacity of the recombinant phage onto DC3000, thereby confirming the functionality of the replaced tail filament. **<Infection Level>** Green fluorescence is used as the forward screening marker. The packaging plasmid pUC19-pac\_J23119 containing the fluorescent protein bjGFP expression cassette was introduced into *Escherichia coli* MG1655 (pUC19-VO98\_215) and *Escherichia coli* MG1655 (pUC19-VO98\_215(C-)) to construct dual-plasmid host strains: *Escherichia coli* MG1655 (pUC19-VO98\_215, pUC19-pac-J23119-GFP) and *Escherichia coli* MG1655 (pUC19-VO98\_215(C-), pUC19-pac-J23119-GFP). Infection of the dual-plasmid hosts with wild-type T7 phage yielded phage particles with capsids containing only the pUC19-pac-J23119-GFP plasmid, where the tail protein had been replaced. DC3000 liquid culture was inoculated with the phage particles, mixed, and spread onto the resistance plates to indirectly verify the infectivity of the recombinant phage against DC3000. ![Fig. 17](https://static.igem.wiki/teams/5846/wiki/engineering/fig-17.webp) **Figure 17: The model of packaging plasmid pUC19-pac-J23119-GFP** **Test** **<DNA Level>** Genomic PCR results for all 4 specific primer pairs matched expectations: * Primer VO98\_215-up\_test showed a band only in T7 ∆gp17::VO98\_215. * Other primers produced bands in all strains except wild-type T7, with correct band sizes. Sequencing results confirmed the presence of phages with successful genomic recombination and gene replacement in the recombinant progeny. ![Fig. 18](https://static.igem.wiki/teams/5846/wiki/engineering/f18.avif) **Figure 18: The results of phage genome PCR.** For primers VO98\_215-up\_test and VO98\_215-down\_test, the expected positive result band size is approximately 200bp. For primer VO98\_215/(C-)-up, the expected positive result band size is approximately 700bp. For primer VO98\_215/(C-)-up, the expected positive result band size is approximately 1400bp. **<Protein Level>** Wild-type T7 supernatant showed no bands, while T7 ∆gp17::VO98\_215 and T7 ∆C-gp17::C-VO98\_215 exhibited bands, confirming successful expression of the new tail filament protein following gene replacement. ![Fig. 19](https://static.igem.wiki/teams/5846/wiki/engineering/f19.avif) **Figure 19: Western Blot of phage WT-T7, T7 ∆gp17::VO98\_215 and T7 ∆C-gp17::C-VO98\_215.** The expected band size of the target protein is approximately 64 kDa, and it is present only in the phage T7 ∆gp17::VO98\_215 and T7 ∆C-gp17::C-VO98\_215. **<Adsorption Level>** Wild-type T7 phages only approached DC3000 cells and exhibited negligible adsorption. Recombinant phages T7 ∆gp17::VO98\_215 and T7 ∆C-gp17::C-VO98\_215 showed varying degrees of adsorption to DC3000 cells. ![Fig. 20](https://static.igem.wiki/teams/5846/wiki/engineering/f20.avif) **Figure 20: Negative staining Transmission Electron Microscope detects the ability of phage with recombinant tail fiber to infect Pseudomonas syringae pv. *Tomato* DC3000.** A: Dividing *Pseudomonas syringae pv. Tomato* DC3000; B: *Pseudomonas syringae pv. Tomato* DC3000; C: T7 bacteriophage; D: WT-T7 can approach but cannot infect *Pseudomonas syringae pv. Tomato* DC3000; E: T7 ∆gp17:::VO98\_215 infecting *Pseudomonas syringae pv. Tomato* DC3000; F: T7 ∆C-gp17:: C-VO98\_215 infecting *Pseudomonas syringae pv. Tomato* DC3000. **<Infection Level>** Neither the recombinant phage nor the wild-type T7 phage could lyse DC3000, thus phage plaques were not directly observable. However, phage-like particles with genomes replaced by the packaging plasmid and altered tail filaments injected the packaging plasmid into DC3000 cells upon infection. After plating, these particles produced kanamycin-resistant DC3000 colonies that exhibited green fluorescence. ![Fig. 21](https://static.igem.wiki/teams/5846/wiki/engineering/fig-21.webp) **Figure 21: LB plates spread with DC3000 after infection with PLPs** **Learn** Experiments confirm successful tail replacement and demonstrate that tail-modified phage-like particles effectively infect target bacteria. These particles, which successfully encapsulate the packaging plasmid with altered tails, successfully delivered the plasmid from *E. coli* to DC3000. This confirms that the phage-like particles we obtained can deliver DNA from the chassis organism to the target organism. **3.3 Construction of pUC19\_VO98\_215（C-）****1st Iteration****Design** A recombinant fragment containing the gene replacement homologous arm and the vector homologous arm was obtained via nested PCR from the pUC19\_VO98\_215 vector. After linearizing the pUC19 vector with the restriction enzyme EcoRI, the recombinant fragment was seamlessly cloned into the vector. **Build** Using vector pUC19-VO98\_215 as a template, the recombinant fragment was obtained through two rounds of PCR. After the first round, the fragment contained the complete downstream homologous arm (*gp17* down arm) of the gene replacement and a partial upstream homologous arm (*gp17*(149) up arm). The second round of PCR completed the upstream homologous arm and added vector homologous arms to both ends of the fragment. The pUC19 vector was digested with the restriction enzyme EcoRI and verified by electrophoresis. **Test** The seamless cloning product was transformed into MG1655 and plated. However, multiple colony PCRs failed to yield the expected positive clones. Notably, the negative control plate showed numerous colonies, indicating a high blank transformation rate. ![Fig. 22](https://static.igem.wiki/teams/5846/wiki/engineering/fig-22.webp) **Figure 22: The results of colony PCR. Positive colonies were expected at around 2kb and there are no colonies in the figure that match the expectation.** ![Fig. 23](https://static.igem.wiki/teams/5846/wiki/engineering/fig-23.webp) **Figure 23: Negative Control Plate.** **Learn** Upon careful analysis, we concluded that the root cause lay in the challenging control of the ratio between the recombinant fragment and the linearized pUC19 vector. Additionally, the small size of the pUC19 vector led to a high rate of vector self-ligation and linear fragment transfer during transformation. Furthermore, EcoRI digestion could not guarantee complete linearization of all pUC19 vectors, contributing to the failure to obtain positive clones. **2nd Iteration****Design** To address the high blank rate, we decided to obtain the linearized vector using reverse PCR followed by DpnI treatment combined with gel recovery. **Build** High-purity linearized vector was obtained via reverse PCR, DpnI digestion, and gel recovery, with all other procedures unchanged. **Test** Colony PCR confirmed the desired positive clones. ![Fig. 24](https://static.igem.wiki/teams/5846/wiki/engineering/fig-24.webp) **Figure 24: The results of colony PCR. Positive colonies were expected at around 2kb, which two picked colonies in the lane 3-4 turned out to be.** **Learn** Blank transformations remain a significant challenge in seamless cloning vector construction. Employing reverse PCR to generate linearized vectors followed by DpnI digestion proves an effective strategy for enhancing positive clone yield. Dry LabOverview Learn how we developed Alphage — an intelligent tool for precisely modifying T7 phage tail-fiber proteins — through iterative engineering cycles, and see how we systematically enhanced its scientific rigor and user-friendliness. Software1st IterationDesign To predict phage tail fiber modification schemes, we developed Alphage, a tool designed to assist the Wet Lab in identifying replacement sites for tail fiber replacement. We relied on homology to identify replacement sites and defined the UI logic. Build

We first performed pairwise global alignment of the two tail-fiber sequences to compute homology and assess each site’ s replaceability. Concurrently, a simple UI was designed for testing purposes.

![Figure 25](https://static.igem.wiki/teams/5846/wiki/engineering/fig-25.webp)

**Figure 25: Picture of the initial software interface**

#### Test Preliminary testing revealed that the software generated only a limited number of valid replacement sites. After discussion, we concluded that the simplistic sequence alignment logic would miss numerous potential homologous fragments. ![Figure 26](https://static.igem.wiki/teams/5846/wiki/engineering/fig-26.webp) **Figure 26: Picture of the initial result graph** Learn We need to adopt a more comprehensive sequence alignment tool with in-depth computational capabilities to ensure full-sequence alignment as thoroughly as possible. 2nd IterationDesign In this cycle, we selected DeepBLAST — a sequence alignment tool based on convolutional neural networks (CNNs), which demonstrates high accuracy in aligning distantly related sequences. Build We deployed DeepBLAST on a local server and tail-fiber amino-acid sequences from the initial database were aligned against the T7 tail-fiber reference. Test DeepBLAST’ s full-sequence alignment function is robust, adequately meeting our needs for assessing the homology of tail fiber protein sequences. However, the existing software lacked the ability to evaluate the feasibility of successful replacement at replacement sites. Learn Next, we need to explore alternative approaches to assess the reliability of replacement sites. 3rd IterationDesign In this cycle, through discussions and consultations with instructors and professors, we selected ESMFold for structure-based stability estimation and PHIEmbed for phage–host interaction prediction. Build We integrated PHIEmbed into the software to predict the efficiency of phage-host infection after replacing the T7 phage tail fiber protein at the replacement site. In parallel, we consulted Prof. Yue Feng (Beijing University of Chemical Technology), who recommended incorporating stability assessment. Consequently, ESMFold was integrated to generate pLDDT scores as a proxy for structural integrity. Test Currently, the three models in the software work well together and generate biologically meaningful parameters during computation. However, these parameters lack interconnections to support the overall evaluation of tail fiber protein replacement schemes. Learn Based on the existing indicator scores, we need to achieve objective integration of multi-dimensional scores and construct a more scientifically robust comprehensive scoring model. 4th IterationDesign To achieve objective integration of multi-dimensional scores, we used the “paired ranking transformation + linear regression optimized by binary cross-entropy” algorithm to solve for the optimal weight coefficients among the scores. Burges, C., Shaked, T., Renshaw, E., Lazier, A., Deeds, M., Hamilton, N., & Hullender, G. (2005). Learning to rank using gradient descent. In Proceedings of the 22nd International Conference on Machine Learning (pp. 89–96). ACM. Build We collected the currently available scoring datasets and constructed a robust machine learning model. Using the “paired ranking transformation + linear regression optimized by binary cross-entropy” algorithm, we solved for the optimal weight coefficients among the scores, ensuring the weight assignment is statistically valid and supported by data. Test The model provided default weights for the evaluation parameters: 62% for “homology”, 29% for “Δhomology”, and 9% for “infection”. Based on these weights, comprehensive scores were computed, with schemes ranked in descending order of confidence. ![Figure 27](https://static.igem.wiki/teams/5846/wiki/engineering/fig-27.webp) **Figure 27: Parameters setting interface** Learn So far, the software’ s computational logic is essentially complete; what we still need is an appealing UI to deliver a more intuitive and comfortable user experience. For more information of our final results, please visit our [wiki page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27software%27%29%20%7D%7D). 5th IterationDesign To deliver a better user experience, we have redesigned the UI based on the software’ s computational logic. Build We added an interactive interface allowing users to adjust parameter weights. On the results page, the computed replacement schemes are presented through line graphs paired with concise textual descriptions. Test We conducted tests using the project’ s replacement schemes and obtained the following results: ![Figure 4](https://static.igem.wiki/teams/5846/wiki/engineering/fig-28.webp) **Figure 28: Result display interface** Learn Currently, we have successfully developed Alphage, a robust tool for predicting phage tail fiber protein replacement. In the future, we will further expand the existing dataset and conduct comprehensive testing of the software’ s functions. Human PracticeOverview To enhance the effectiveness of synthetic biology education, we iteratively developed a **story-based curriculum** for primary students and a card game using the “Design-Build-Test-Learn” cycle. The CHUN GENG Program built a logic-driven knowledge system using metaphors and character guidance, while SynBio Halli Galli simplified component recognition with color-coded pathways. Both projects evolved through user feedback, transitioning from concept delivery to systemic understanding, demonstrating the flexibility and impact of engineering thinking in science communication. SynBio Halli Galli Engineering1st IterationDesign In the initial phase, we set the popular science objective as enabling players to memorize synthetic biology components and understand the construction of genetic pathways. Based on the characteristics of this objective, we drew inspiration from the game Halli Galli and adopted its following advantages: Build We defined three types of cards: * **Component Cards**: These include four basic synthetic biology components. * **Contamination Cards**: These simulate laboratory biosafety scenarios such as phage outbreaks, culture medium contamination, and mutation accumulation, with an emphasis on **biosafety**. * **Function Cards**: These introduce commonly used tools like CRISPR-Cas9 and DNA ligase. Additionally, we retained the basic rules of Halli Galli including reward and penalty mechanisms as well as the method for determining victory or defeat. Test We contacted team members who worked on the previous card game project, and we **jointly discussed the playability and rationality of our cards**. Their feedback was: a single pathway might bore players, adding diverse promoters and designing pathways with different functions would help increase the diversity of the cards. Learn We incorporated their suggestions, leading to **several key changes** in our card design: 1. We redesigned three types of promoters: a promoter that responds extensively to low pH, an anaerobically activated promoter, and a heat-induced promoter. 2. Inspired by these multiple promoters, we optimized the target genes for the corresponding conditions: fluorescent protein genes, drug resistance genes, and protease genes. 2nd IterationDesign Based on the feedback from the first iteration, we began designing the component patterns to be displayed on the cards. Build We aimed for the component patterns to **intuitively represent the conditions for pathway expression** while being both **aesthetically pleasing and concise**. With this in mind, we created designs for each component card, contamination card, and tool card. Test Before officially producing the card designs, we **hand-drew** 90 cards using pencils and cut paper to make physical prototypes, then invited other team members to **test-play** them. From the test-play experience, we identified a problem in the design: when components with overly detailed drawings were arranged in quantities ranging from 1 to 4 on a 6×8 cm card, **the detailed patterns became too small**. This significantly increased the game difficulty, prolonged reaction times (which slowed down the game pace), and failed to effectively stimulate players’ reaction abilities. Learn Therefore, we began to consider: how to **maximize simplification** while retaining the key features of the components. As a result, we chose to keep only the symbols and letter abbreviations of the components. For contamination cards and function cards, which could have designs covering the entire card surface, we retained the detailed drawings. 3rd IterationDesign We used different solid colors to fill each type of component, aiming to reduce the game difficulty for players without a background in synthetic biology. When constructing genetic pathways, players could **use the color guidance to gain a clearer understanding of the complete pathway structure**, thereby achieving our popular science goal. Build In accordance with our design plan, we added colors to the original hand-drawn patterns and launched **the second round of test-playing**. Test With the experience from the first test-play, we became much more proficient in the second round. However, after several rounds, we gradually noticed that the construction of **a single pathway was somewhat monotonous.** Learn Thus, based on biological knowledge, we **distinguished between eukaryotic and prokaryotic genetic pathways**, using solid and hollow drawing styles respectively to differentiate them. At this point, our card game was finalized. We finalized the card designs, commissioned the production of a certain number of physical cards, and released the card instruction manual via a promotional post. The cards became a key element in scenarios such as popular science education, team communication, and project demonstrations. CHUN GENG Program Engineering1st IterationDesign We planned to expand the audience of synthetic biology popular science to **primary school students**, creating an **immersive learning experience** by combining games and stories. Considering the natural appeal of picture books to children, we decided to create **a character named “Chocolate”** (based on a real teddy bear dog named Chocolate). **Through Chocolate’ s perspective to connect scattered knowledge points**, we would guide children into the world of synthetic biology. During the popular science classes, we would **intersperse game activity scenarios as one form of immersive popular science**, creating an atmosphere where the character and the students explore synthetic biology together, and realize the game through **character interaction**. Build We built a **story framework**: the dog Chocolate travels to the world of synthetic biology and explores the mysteries of this world with children. We created a 5-minute PPT story, **anthropomorphizing cells and endowing them with different “job functions”**, and adapted previous iGEM projects into plotlines. In the story, DNA is compared to a “cell instruction manual”, bases to “letters”, biological components to “words”, biological devices to “sentences”, and genetic pathways to “instructions”. Test We **conducted a trial lecture** for students with no background in synthetic biology. The participants had a good understanding of the metaphors in the story and particularly **resonated with the character** “Chocolate”. They were able to retell the metaphors in the story and initially understand “how cells work”, **which confirmed the feasibility of story-based popular science**. Learn We verified that the **“storytelling + character guidance”** form of popular science is effective for primary school students. On this basis, we decided to further expand the content, adopting the **“language system”** analogy method to gradually build **a complete knowledge system**. Based on the story framework, we continued to expand the popular science content, using the language system familiar to children (DNA - cell instruction manual, bases - letters, biological components - words, biological devices - sentences, genetic pathways - instructions guiding cell work) to analogize synthetic biology concepts, and organized the teaching content in accordance with the **bottom-up thinking of engineering**. 2nd IterationDesign Building on the success of the first iteration, we expanded the 5-minute story into a complete **90-minute course**, covering the entire process **from basic concepts to logical structures**. Build The story still starts with specific synthetic biology examples. Under the framework of the language system, it introduces the material basis (compared to **writing materials**) from the most basic level, the central dogma (**grammar**), and the previous synthetic biology concepts. Moreover, the concepts of feedforward, positive feedback, and negative feedback are introduced into genetic circuits, which are analogized through the protagonist **“Chocolate’ s” three twists and turns of experiences in wanting to eat ice cream**, to **compare the connections and differences between the three circuits**. For example: * **Feedforward structure**: Finish homework first before making a request to increase the success rate; * **Positive feedback**: Moderate behavior leads to long-term trust; * **Negative feedback**: Excessive behavior leads to restrictions. Test Before the official class on the next day, other members of the CHUN GENG Program attended a trial class, **fully simulating the teaching scenario of the next day**. Regarding the complete story Chocolate’ s Journey into Synthetic Biology, some students pointed out that it was somewhat **redundant to explain the concepts in plain language first and then present the concepts themselves**; it would be better to directly replace the concepts with the metaphorical terms. Learn This suggestion led to **a major adjustment in the focus of our class**. We realized that for primary school students, understanding logic is more important than memorizing concepts. **We weighed the pros and cons of presenting concepts and weakening concept indoctrination.** First, too many concepts might make it too difficult for primary school children to understand; on the other hand, weakening concept indoctrination might affect children’ s accurate perception of synthetic biology. **We spent a long time weighing and making a decision**. 3rd IterationDesign Based on the second iteration, **we finally completely replaced professional concepts with metaphorical language**. We clarified that the focus of our popular science class lies in **whether children can fully understand the logic and principles behind synthetic biology.** The memorization of concepts, based on understanding, will become easy, and the timing (sooner or later) does not matter. Build As a result, we reduced concept indoctrination and made the following replacements: * Use **“cell instruction manual”** instead of “DNA”; * Use **“letters”** instead of “bases”; * Use **“words”** instead of “biological components”; * Use **“sentences”** instead of “biological devices”; * Use **“instructions”** instead of “genetic pathways”. Test In the official class, considering the difficulty of the course, we designed a rich **reward mechanism** in the class to encourage children: small stars were given out based on the number of correct answers, and different rewards were given at different levels according to the total number of stars accumulated; in addition, after every 2-3 knowledge points, we set up **small question challenges**, and the questions were completely designed based on the previous knowledge points. Learn **The class received a positive response.** Based on the children’ s performance in the question challenges and the accuracy of their answers, the children had a good grasp of the knowledge. In the final activity of “designing a life instruction manual”, **they were able to independently combine “words” and “sentences” to construct simple genetic circuits**. This activity not only improved the children’ s thinking ability and broadened their cognitive horizons but also made them feel the fun and magic of learning synthetic biology. Through three iterations, the CHUN GENG Program gradually moved **from “story guidance” to “logical understanding”**, and **from “terminology indoctrination” to “metaphor construction”**. With this, **we attempted to build a synthetic biology popular science system suitable for primary school students**, with characters as the main line and the language system as the framework. The entire process realized the **effectiveness and flexibility** of the “Design - Build - Test - Learn” cycle. References [1] Hamamsy, T., Morton, J.T., Blackwell, R. et al. Protein remote homology detection and structural alignment using deep learning. Nat Biotechnol 42, 975–985 (2024). https://doi.org/10.1038/s41587-023-01917-2 [2] Zeming Lin et al., Evolutionary-scale prediction of atomic-level protein structure with a language model. Science 379, 1123–1130 (2023). DOI: 10.1126/science.ade2574 [3] Gonzales MEM, Ureta JC, Shrestha AMS. Protein embeddings improve phage-host interaction prediction. PLoS One. 2023 Jul 24;18(7):e0289030. doi: 10.1371/journal.pone.0289030. PMID: 37486915; PMCID: PMC10365317

{% endblock %}

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{% block title %}Experiments{% endblock %}
{% block lead %}https://static.igem.wiki/teams/5846/wiki/header-image/experiments.webp{% endblock %}
{% block nav\_text\_1 %}▶ Preparation of Basic Experimental Material{% endblock %}
{% block nav\_text\_1\_1 %}◆ LB{% endblock %}
{% block nav\_text\_1\_2 %}◆ LB Agar{% endblock %}
{% block nav\_text\_1\_3 %}◆ Preparation of LB-Antibiotics{% endblock %}
{% block nav\_text\_1\_4 %}◆ Preparation of Agarose Gel{% endblock %}
{% block nav\_text\_1\_5 %}◆ TY Medium{% endblock %}
{% block nav\_text\_1\_6 %}◆ TY Agar{% endblock %}
{% block nav\_text\_2 %}▶ Molecular Biology Experiment{% endblock %}
{% block nav\_text\_2\_1 %}◆ Genome Extraction{% endblock %}
{% block nav\_text\_2\_2 %}◆ PCR{% endblock %}
{% block nav\_text\_2\_3 %}◆ Plasmid Extraction{% endblock %}
{% block nav\_text\_2\_4 %}◆ Plasmid Digestion{% endblock %}
{% block nav\_text\_2\_5 %}◆ Agarose Gel Electrophoresis (AGE){% endblock %}
{% block nav\_text\_2\_6 %}◆ DNA Extraction from Agarose Gel{% endblock %}
{% block nav\_text\_2\_7 %}◆ Gibson Assembly{% endblock %}
{% block nav\_text\_3 %}▶ Microbiology Experiment{% endblock %}
{% block nav\_text\_3\_1 %}◆ Transformation of chemically competent *E. coli*{% endblock %}
{% block nav\_text\_3\_2 %}◆ General bacterial concentration testing{% endblock %}
{% block nav\_text\_3\_3 %}◆ Bacterial plating{% endblock %}
{% block nav\_text\_3\_4 %}◆ Phenotype inducing{% endblock %}
{% block nav\_text\_4 %}▶ Agarose Gel Electrophoresis Analysis{% endblock %}
{% block nav\_text\_4\_1 %}◆ Methods{% endblock %}
{% block nav\_text\_5 %}{% endblock %}
{% block nav\_text\_6 %}{% endblock %}
{% block nav\_text\_7 %}{% endblock %}
{% block nav\_text\_8 %}{% endblock %}
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# Preparation of Basic Experimental Material

## 1. LB

### Materials:

* Tryptone
* Yeast extract
* NaCl AR
* dH2O
* Conical flask
* Microwave
* Magnetic stirrer and rotor

### Protocol:

1. Weigh reagents in the following proportions:

| Substance | Concentration |
| --- | --- |
| Tryptone | 10 g/L |
| Yeast extract | 5 g/L |
| NaCl | 10 g/L |

2. Deposit them all into the flask.
3. Add distilled water to the expected volume.
4. Stir the mix until the powders are completely dissolved.
5. Autoclave for 20 minutes at 121°C.
6. Store at 4°C.

---

## 2. LB Agar

### Materials:

* Tryptone
* Yeast extract
* NaCl AR
* Agar
* dH2O
* Conical flask
* Microwave
* Magnetic stirrer and rotor

### Protocol:

1. Weigh reagents in the following proportions:

| Substance | Concentration |
| --- | --- |
| Tryptone | 10 g/L |
| Yeast extract | 5 g/L |
| NaCl | 10 g/L |

2. Deposit them all into the flask.
3. Add distilled water to the expected volume.
4. Stir the mix until the powders are completely dissolved.
5. Add 1.5% weight agar into the flask.
6. Autoclave for 20 minutes at 121°C.
7. Store at 4°C.

---

## 3. Preparation of LB-Antibiotics

* If the medium does not contain agar: Add one thousandth of a volume of antibiotic solution, mix thoroughly.
* If the medium does contain agar: When the temperature is reduced to approximately 45°C, add one thousandth of a volume of antibiotic solution, then mix it thoroughly. After antibiotics are added, it should not be heated again.

---

## 4. Preparation of Agarose Gel

### Materials:

* Agarose
* 1x TAE
* Graduated cylinder
* Conical flask
* Pipettor and pipette
* Microwave
* 10,000x nucleic acid dye

### Protocol:

1. Add the exact weight of agarose to the measuring cylinder to ensure the correct concentration.
2. Add 1x TAE of the required volume.
3. Heat the solution in the microwave until it boils.
4. Take the cylinder out and let it cool down until it can be touched by hands.
5. Add nucleic acid dye that is 1/10,000 of the total volume of the system.
6. Pour into electrophoresis box, place the comb, and wait until it solidifies.
7. Take out the comb carefully.

---

## 5. TY Medium

### Materials:

* Tryptone
* Yeast extract
* CaCl2 AR
* dH2O
* Conical flask
* Microwave
* Magnetic stirrer and rotor

### Protocol:

| Substance | Concentration |
| --- | --- |
| Tryptone | 5 g/L |
| Yeast extract | 3 g/L |
| CaCl2 | 0.6 g/L |

1. Deposit them all into the flask.
2. Add distilled water to the expected volume.
3. Stir the mix until the powders are completely dissolved.
4. Autoclave for 20 minutes at 121°C.
5. Store at 4°C.

---

## 6. TY Agar

### Materials:

* Tryptone
* Yeast extract
* CaCl2 AR
* Agar
* dH2O
* Conical flask
* Microwave
* Magnetic stirrer and rotor

### Protocol:

| Substance | Concentration |
| --- | --- |
| Tryptone | 5 g/L |
| Yeast extract | 3 g/L |
| CaCl2 | 0.6 g/L |

1. Deposit them all into the flask.
2. Add distilled water to the expected volume.
3. Stir the mix until the powders are completely dissolved.
4. Add 2% weight agar into the flask.
5. Autoclave for 20 minutes at 121°C.
6. Store at 4°C.

If needed, 5% sucrose can be added to TY or TY agar.

---

# Molecular Biology Experiment

## 1. Genome Extraction

### Materials:

1. Centrifuge tubes (1.5 mL & 2.0 mL)
2. G+ Bacteria Genomic DNA Kit (Zomanbio)
3. RNase A (10 mg/mL) (Zomanbio)
4. ddH2O
5. Ethanol
6. Centrifugal machine
7. Vortex machine
8. Water bath

### Protocol:

1. Centrifugate 1-5 mL bacteria solution for 1 min at 13,400 ×g to retain the pellet.
2. Add 500 μl cell suspension solution (with Lysozyme added) to the bacterial pellet, vortex until the pellet is re-suspended. Incubate at 37℃ for 30 minutes, inverting every 5-10 minutes. Centrifuge at 13,400 ×g for 2 minutes and thoroughly remove the supernatant.
3. Add 225 μl Buffer A and mix thoroughly.
4. Add 6 μl RNase A (10 mg/mL) solution, vortex for 5 seconds and mix well.
5. Add 25 μl lysis solution S and mix thoroughly. Incubate at 56℃ for 20 minutes, inverting 1-2 times during this period.
6. Add 250 μl Buffer B, vortex for 5 seconds and mix thoroughly.
7. Add 250 μl ethanol, vortex for 15 seconds. Transfer the mixture to the column and place the column in the collection tube.
8. Centrifuge at 13,400 ×g for 30 seconds, discard the supernatant. Place the absorption column in the collection tube.
9. Add 500 μl Buffer C, centrifuge at 13,400 ×g for 30 seconds, discard the supernatant, and place the column back into the collection tube.
10. Add 600 μl Washing Solution W2 (with ethanol added), centrifuge at 13,400 ×g for 30 seconds, discard the supernatant, and place the column back into the collection tube.
11. Add an additional 500 μl Washing Solution W2 (with ethanol added), centrifuge at 13,400 ×g for 30 seconds, discard the supernatant, and place the column back into the collection tube.
12. Centrifuge for 2 minutes at 13,400 ×g. Open the column and let it sit at room temperature for a few minutes.
13. Place the column in a new 1.5 mL centrifuge tube, add 100-200 μl elution buffer TE, and let it sit at room temperature for 2-5 minutes.
14. Centrifuge for 2 minutes at 13,400 ×g. Transfer the liquid in the collection tube to the column.
15. Dispose of the column and store the eluate at -20℃.

---

## 2. PCR

### 2.1 General PCR

**Goal:** Amplify a gene or add homology arms to a sequence.

#### Materials:

* PCR tubes (200 μl)
* GoldenStar T6 Super PCR Mix (1.1x)
* Forward primer (10 μM)
* Reverse primer (10 μM)
* Template
* Thermocycler

#### Protocol:

* Add in a PCR tube:

| Substance | Volume (system 25μl/50μl) |
| --- | --- |
| Golden Mix | (23-x) μl/(46-x) μl |
| Forward primer (10 μM) | 1 μl/2 μl |
| Reverse primer (10 μM) | 1 μl/2 μl |
| Template | x μl (depends) |

* Place the tube in the thermocycler and start corresponding program.
* After the sample temperature decreases to 4℃, store the result at -20℃.

### 2.2 Fusion PCR

**Goal:** Connect two DNA fragments containing homologous arms together.

#### Materials:

1. PCR tubes (200 μl)
2. GoldenStar T6 Super PCR Mix (1.1x)
3. Template (containing homologous arms)
4. Forward primer (10 μM)
5. Reverse primer (10 μM)
6. Thermocycler

#### Protocol:

* Add in a PCR tube:

| Substance | Volume (system 25μl/50μl) |
| --- | --- |
| Golden Mix | (23-x-y) μl/(46-x-y) μl |
| Template1 | x μl (depends) |
| Template2 | y μl (depends) |

* Add 1 μl/2 μl of each of the two primers.
* Place the tube in the thermocycler and start corresponding program.
* After the sample temperature decreases to 4℃, store the result at -20℃.

### 2.3 Colony PCR

**Goal:** To determine whether a certain sequence exists in the thallus.

#### Materials:

1. PCR tubes (200 μl)
2. 2 x Taq Master Mix (dye) / 2 x T5 Super PCR Mix (Basic)
3. Forward primer (10 μM)
4. Reverse primer (10 μM)
5. ddH2O
6. Thermocycler

#### Protocol:

1. Add in a PCR tube:

| Substance | Volume (system 25μl) |
| --- | --- |
| 2 x Taq Master Mix / 2 x T5 Super | 12.5 μl |
| Forward primer (10 μM) | 1 μl |
| Reverse primer (10 μM) | 1 μl |
| ddH2O | 10.5 μl |

2. Usually, we configure more premix than the required number of systems in a 1.5 mL centrifuge tube, then separate them into PCR tubes.
3. Place the tube in the thermocycler and start corresponding program.
4. After the sample temperature decreases to 4℃, stop the program and use the results for AGE detection.

---

## 3. Plasmid Extraction

### 3.1 Zomanbio

#### Materials:

* Centrifuge tubes (1.5 mL & 2.0 mL)
* Fast Plasmid Miniprep Kit (Zomanbio)
* Centrifugal machine
* Vortex machine

#### Protocol:

1. Centrifugate 1-5 mL bacteria solution 1 min at 13,400 x g, retain precipitation.
2. Add 250 μl Buffer K1 (RNase added) in the bacteria precipitation, vortex it until the thallus is resuspended.
3. Add 250 μl buffer K2, gently mix it until the liquid looks clear.
4. Add 350 μl buffer K3, immediately mix it gently.
5. Centrifugate the tube 2 min at 13,400 ×g, transfer the supernatant to column and put it in the collection tube, centrifugate 30-60 s at 13,400 ×g. Discard supernatant, replace the column into the collection tube.
6. If the bacteria is *E. coli* BL21 (DE3) or Pseudomonas fluorescens 2P24, add 500 μl Deproteinized solution W1 in column and centrifugate 30-60 s at 13,400 ×g. If the bacteria is *E. coli* DH5 α, skip this step.
7. Add 500-600 μl Washing Solution W2 (ethanol added) in column and centrifugate 20 s at 13,400 ×g. Repeat this step.
8. Centrifugate 1 min at 13,400 ×g.
9. Put the column in a new centrifuge tube, add 80-150 μl elution buffer TE. Place it for 1 min.
10. Centrifugate 1 min at 13,400 ×g. Discard the column and store the result at -20℃.

### 3.2 TransGen

#### Materials:

* Centrifuge tubes (1.5 mL & 2.0 mL)
* EasyPure Plasmid Miniprep Kit (TransGen)
* Centrifugal machine
* Vortex machine

#### Protocol:

1. Before use, RNase A was added to RB and stored at 2-8℃. Add different volumes of 100% ethanol to WB.

| LB Media Volume | RB (μl) | LB (μl) | NB (μl) |
| --- | --- | --- | --- |
| 5 mL | 250 | 250 | 350 |
| 5-10 mL | 500 | 500 | 700 |
| 10-15 mL | 750 | 750 | 1,050 |
| 15-20 mL | 1,000 | 1,000 | 1,400 |

1. Take the overnight culture, centrifuge at 10,000 ×g for 1 minute, and remove the supernatant.
2. According to the table, add colorless solution RB (containing RNase A), vortex to resuspend the bacterial pellet, ensuring no small clumps remain.
3. According to the table, add blue solution LB, gently invert 4-6 times to mix, ensuring complete lysis of the bacteria, forming a blue and clear solution. The color change from semi-transparent to clear blue indicates complete lysis.
4. According to the table, add yellow solution NB, gently mix 5-6 times (the color change from blue to yellow indicates complete mixing and neutralization), until a compact yellow precipitate forms, then let it stand at room temperature for 2 min.
5. Centrifuge at 12,000 ×g for 5 min, carefully transfer the supernatant to a centrifuge column. Centrifuge at 12,000 ×g for 1 min, discard the flow-through.
6. Add 650 μl solution WB, centrifuge at 12,000 ×g for 1 min, and discard the flow-through.
7. Centrifuge at 12,000 ×g for 1-2 min to thoroughly remove residual WB.
8. Place the centrifuge column in a clean centrifuge tube, add 20 μl EB to the center of the column, and let it stand at room temperature for 1 min. (Pre-warming EB in a 60-70°C water bath improves the outcome).
9. Centrifuge at 10,000 ×g for 1 min to elute the DNA, and store the eluted DNA at -20°C.

### 3.3 Mei5bio

#### Materials:

* Centrifuge tubes (1.5 mL & 2.0 mL)
* M5 HiPer Multi-color Plasmid Miniprep Kit (Mei5bio)
* Centrifugal machine
* Vortex machine

#### Protocol:

1. Column Equilibration: Add 400 μL of Buffer BL to the centrifugal adsorption column, let it stand for 1 minute, centrifuge at 12,000 rpm at room temperature for 1 minute, discard the waste liquid in the collection tube, and place the centrifugal adsorption column back into the collection tube.
2. Take 1-5 mL of overnight culture, centrifuge at 12,000 rpm at room temperature for 1 minute, and remove the supernatant as completely as possible.
3. Add 250 μL of Solution I, vortex or pipette thoroughly to resuspend the bacteria evenly, resulting in a uniformly turbid brown-red color.
4. Add 250 μL of Solution II, gently invert to mix well, allowing the bacteria to fully lyse until the solution becomes clear and viscous purple-red.
5. Add 350 μL of Solution III, immediately and gently invert to mix, a precipitate with red and yellow layers will be visible, continue mixing until it turns completely yellow, let it stand at room temperature for 2 min, then centrifuge at 12,000 rpm for 5 min.
6. Carefully transfer the supernatant to the centrifugal adsorption column, let it stand for 2 min to allow the plasmid to bind fully with the silica membrane of the column. Centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate in the collection tube.
7. Add 500 μL of Buffer WB2, centrifuge at 12,000 rpm at room temperature for 30 seconds, and discard the filtrate in the collection tube. Repeat this step once.
8. Centrifuge at 12,000 rpm at room temperature for 2 min to dry the residual liquid.
9. Place the centrifugal adsorption column in a new 1.5 mL plastic centrifuge tube (provided by the user), add 50-100 μL of elution buffer EB, let it stand at room temperature for 2 min.
10. Centrifuge at 12,000 rpm for 1 minute, and the solution at the bottom of the centrifuge tube is the plasmid DNA. Store the eluted DNA at -20℃.

---

## 4. Plasmid Digestion

**Goal:** To cleave a vector using restriction enzymes to prepare them for ligation.

### Materials:

* rCutSmart Buffer (10x)
* ddH2O
* 200 μL centrifuge tube
* Water bath at 37℃
* Water bath at 65℃

### Protocol:

| Substance | Volume |
| --- | --- |
| Enzymes | 0.1 μl |
| Enzymes | 0.1 μl |
| Plasmid | x μl (depends) |
| rCutSmart Buffer (10x) | 1 μl |
| ddH2O | (8.8-x) μl |

1. Keep warm in a water bath at 37℃ for 1 hour.
2. Keep warm in a water bath at 65℃ for 20 minutes.
3. Correct bands were extracted after electrophoresis.

### Materials:

* 10X QuickCut Green Buffer
* ddH2O
* 200 μL centrifuge tube
* Water bath at 37℃
* Water bath at 80℃

### Protocol:

| Substance | Volume |
| --- | --- |
| Enzymes | 1 μl |
| Plasmid | x μl (depends) |
| 10X QuickCut Green Buffer | 1 μl |
| ddH2O | (8-x) μl |

1. Keep warm in a water bath at 37℃ for 1 hour.
2. Keep warm in a water bath at 80℃ for 15 minutes.
3. Correct bands were extracted after electrophoresis.

---

## 5. Agarose Gel Electrophoresis (AGE)

**Goal:** Separating DNA bands according to their length, and estimating the length and DNA concentration of each band. When digested products or recombinant products are subjected to electrophoresis, care should be taken to use the original plasmids for control.

### Materials:

* Ready-made agarose gel
* DNA sample
* 10 x loading buffer (dye)
* DNA Ladder/Marker
* 1 x TAE
* Electrophoresis tank

### Protocol:

1. Place the gel on the gel tray within the electrophoresis system, make sure the wells are located at the negative electrode.
2. Mix loading buffer with the DNA sample if the sample is not coloured. Usually, the loading buffer has one tenth the volume of the sample, but the volume should not be less than 0.3 μl.
3. Load either the DNA ladder or the samples within the wells. Make sure that the sample or ladder sinks to the bottom of the well. The volume should be between 3 μl to 5 μl, depending on the well’ s capacity.
4. Run the gel for 20-40 minutes, depending on the situation. Ensure the voltage is set to 100-150 V.

---

## 6. DNA Extraction from Agarose Gel

### Materials:

* Centrifuge tubes (1.5 mL)
* EasyPure Quick Gel Extraction Kit (TransGen)
* ddH2O
* Centrifugal machine
* Vortex machine
* Water bath

### Protocol:

1. Carefully cut the target band on agarose gel and put it in a centrifuge tube.
2. Add 600 μl GSB. Heat it at 55℃ using a water bath for 6-10 minutes. Mix intermittently to ensure the gel is completely melted.
3. After the gel dissolves, allow the solution to cool to room temperature, transfer the liquid to a column, and put it in the collection tube. Centrifuge for 60 seconds at 10,000 ×g. Discard the flow-through liquid.
4. Add 650 μl WB, centrifuge for 60 seconds at 10,000 ×g. Discard the flow-through liquid.
5. Centrifuge for 1-2 minutes at 10,000 ×g to completely remove residual WB.
6. Place the centrifuge column in a clean centrifuge tube, open the cap and let it stand for 1 minute to allow the residual ethanol to evaporate completely. Then add 20 μl of deionized water (pre-warmed) to the center of the column and let it stand at room temperature for 1 minute.
7. Centrifuge for 60 seconds at 10,000 ×g. Discard the column and store the result at -20℃.

---

## 7. Gibson Assembly

**Goal:** To assemble the different DNA parts retrieved by PCR.

### Materials:

* PCR tubes (200 μl)
* ClonExpress Ultra One Step Cloning Kit
* ddH2O
* Thermocycler

### Protocol:

1. Calculate the required amount of DNA for the recombination reaction according to the formula:

   * Single-fragment: Optimal cloning vector usage = [0.02 x number of base pairs of cloning vector] ng (0.03 pmol) Optimal insert fragment usage = [0.04 x number of base pairs of insert fragment] ng (0.06 pmol)
   * Multi-fragment: Optimal cloning vector usage = [0.02 x number of base pairs of cloning vector] ng (0.03 pmol) Optimal usage for each fragment = [0.02 x number of base pairs of each fragment] ng (0.03 pmol)
2. Prepare the following reaction system on ice:

| Components | Recombination Reaction | Negative Control-1 | Negative Control-2 | Positive Control |
| --- | --- | --- | --- | --- |
| Linearized Vector | X μl | X μl | 0 μl | 1 μl |
| n Insert Fragments | Y1+Y2…+Yn μl | 0 μl | Y1+Y2…+Yn μl | 1 μl |
| 2x ClonExpress Mix | 5 μl | 0 μl | 0 μl | 5 μl |
| ddH2O | to 10 μl | to 10 μl | to 10 μl | to 10 μl |

1. Gently mix well with a pipette (do not vortex mix), briefly centrifuge to collect the reaction liquid at the bottom of the tube.
2. Single-fragment recombination reaction, 50°C, 5 min; cool to 4°C or immediately place on ice.
3. Multi-fragment recombination reaction, 50°C, 15 min; cool to 4°C or immediately place on ice.

---

# Microbiology Experiment

## 1. Transformation of chemically competent *E. coli*

### Materials:

* LB medium
* LB agar plates with antibiotics (depends)
* Crushed ice
* Plasmid used to transform
* Chemically competent cell
* Centrifuge tube (1.5 mL)

### Protocol:

1. Add 100 μl chemically competent cell and 10 μl of plasmid in the tube, mix gently and softly.
2. Incubate the tube for 30 minutes on ice.
3. Incubate for 90 seconds at 42°C, then immediately place on ice for 2 minutes.
4. Add 700-900 μl (depends) of LB liquid medium.
5. Incubate for 45 minutes under agitation at 200 rpm.
6. Centrifuge for 5 minutes at 6,000 ×g.
7. Remove 950 μl of supernatant and resuspend the bacteria with 50 μl of this supernatant.
8. Spread the solution on a LB agar plate containing the relevant antibiotic.
9. Incubate in incubator at 37°C overnight.
10. Check for colonies the next day.

---

## 2. General bacterial concentration testing

### Materials:

* Nano spectrophotometer
* Test solution

### Protocol:

1. Turn on the Nano spectrophotometer and select the measurement mode for bacterial suspension.
2. Calibrate the spectrophotometer with standard samples to ensure accuracy.
3. Add the bacterial suspension to a cuvette and place it in the spectrophotometer to measure the absorbance. Ensure cleanliness and optical path alignment.
4. Record the concentration of the bacterial suspension.

---

## 3. Bacterial plating

### 3.1 Dilution coated plate method

#### Materials:

* Bacteria solution cultured for a period of time
* Solid medium
* Spreader

#### Protocol:

1. Add some bacteria solution onto the surface of the medium.
2. Spread from one side of the plate, draw a few circles, and then overlap the streaks. Use a sterilized spreader and ensure it’ s not too hot.
3. Place the plates in the incubator with clean surfaces. Temperature varies by bacteria type.

### 3.2 Plate streaking

#### Materials:

* Bacteria solution cultured for a period of time
* Solid medium
* Inoculating loop

#### Protocol:

1. Dip inoculating loop into some bacterial liquid.
2. Streak on one side of the plate, do a quarter turn, and then overlap the streaks.
3. Place the plates in the incubator with clean surfaces. Temperature varies by bacteria type.

---

## 4. Phenotype inducing

### Induced by IPTG

1. Culture the bacteria solution for 12 hours, ensuring OD600 is no less than 0.8.
2. Add 16 μl of 50 mg/ml IPTG.
3. After induction, culture at 37°C.
4. Sometimes, a non-induced control group is used.

---

# Agarose Gel Electrophoresis Analysis

1. Gel Preparation (1.5%)
2. Electrophoresis: 3-5 μl of sample, 5 μl of marker, constant voltage of 150V for 30-60 minutes.

---

## Methods

### 1. Magnetic Bead-based Protein Purification

#### 1.1. Buffers Preparation

All purification buffers were prepared with ultrapure water and filtered through 0.22 μm or 0.45 μm membranes prior to use. Two buffer systems were employed for soluble and inclusion body proteins, respectively.

**For Soluble His-Tagged Proteins:**

* Binding/Wash Buffer: 50 mM Tris-HCl, 300 mM NaCl, 20 mM Imidazole, pH 8.0.
* Elution Buffer: 50 mM Tris-HCl, 300 mM NaCl, 300 mM Imidazole, pH 8.0.

**For His-Tagged Proteins from Inclusion Bodies:**

* Binding/Wash Buffer: 50 mM Tris-HCl, 300 mM NaCl, 8 M Urea, pH 8.0.
* Elution Buffer: 50 mM Tris-HCl, 300 mM NaCl, 8 M Urea, 300 mM Imidazole, pH 8.0.

#### 1.2. Sample Preparation

**Bacterial/Yeast Expressed Proteins:**
Cell pellets were resuspended in ice-cold Binding Buffer (1:10-20, w/v) supplemented with 1 mM PMSF, 1% Triton™ X-100, and 1 mM TCEP. Cells were lysed by sonication on ice. The lysate was clarified by centrifugation, and the supernatant was collected for purification.

**Secreted Proteins from Yeast, Insect, or Mammalian Cells:**
Culture supernatant was collected by centrifugation. If necessary, the supernatant was dialyzed against Binding Buffer to remove interfering substances (e.g., EDTA, imidazole, reducing agents) or concentrated via ammonium sulfate precipitation followed by dialysis.

#### 1.3. Magnetic Beads Equilibration

Ni-IDA Magnetic Beads suspension was thoroughly resuspended. An appropriate volume (e.g., 0.2-0.5 mL of 20% suspension per 2 mg target protein) was transferred to a tube. The beads were immobilized using a magnetic separator, and the storage solution was removed. The beads were then washed three times with an equal volume of Binding Buffer.

#### Protocol:

* **Binding:** The clarified sample was incubated with the equilibrated beads for 1 hour at 4°C with gentle mixing.
* **Washing:** The beads were immobilized, and the flow-through was collected. The beads were washed three times with 1 mL of Wash Buffer.
* **Elution:** Bound proteins were eluted by incubating the beads with 1 mL of Elution Buffer for 5 minutes. This step was repeated 2-3 times to maximize recovery.
* **Concentration and Analysis:** The eluate was concentrated using a 30 kDa molecular weight cut-off centrifugal filter unit. Protein purity and yield were analyzed by SDS-PAGE.
* **Regeneration:** Beads were sequentially washed with: 50 mM Tris-HCl (pH 8.0) containing 6 M Guanidine-HCl, deionized water, 0.1% SDS, deionized water, 70% ethanol, deionized water, 100 mM EDTA (pH 8.0), deionized water, and finally recharged with 100 mM NiSO₄ overnight at 15°C with shaking. Regenerated beads were stored in 20% ethanol at 4°C.

---

### 2. Bacteriophage Adsorption and DNA Injection Assay by Transmission Electron Microscopy (TEM)

#### 2.1. Phage-Bacteria Co-incubation

*Lactococcus lactis* subsp. *cremoris* 3107 (wild-type and mutant strains) were grown in GM17 medium to an OD620nm of 0.25 (early exponential phase). Bacteriophage TP901-1 lysates were dialyzed against SM Buffer (50 mM Tris-HCl, 100 mM NaCl, 10 mM MgSO₄, pH 7.5) and adjusted to a titer of approximately 1×10⁸ PFU/mL.
A mixture containing 0.15 mL bacterial culture, 0.15 mL phage lysate, and 0.2 mL of a 1:10 dilution of GM17 broth in quarter-strength Ringer’ s solution was incubated at 25°C for 10 minutes.

#### 2.2. Sample Processing for TEM

The reaction mixture was immediately placed on ice and centrifuged at 12,000 × g for 10 minutes at 4°C. The pellet was washed twice with ice-cold quarter-strength Ringer’ s solution and finally resuspended in 50 μL of the same solution.
Carbon films floated on mica plates were directly applied to the bacteria-phage mixtures for 10 minutes to adsorb the complexes. The specimens were then negatively stained with 1% uranyl acetate (aqueous solution) for a few seconds.
Micrographs were acquired using either an EM 300 or a Tecnai 10 transmission electron microscope (Philips) operating at an acceleration voltage of 80 kV.

---

### 3. Bacteriophage Concentration and Titration

#### 3.1. Phage Concentration by PEG Precipitation

A 200 mL culture of the host bacterium (*Salmonella* strain G33T-2) was grown in LB broth at 37°C with shaking (220 rpm) to an OD600 of ~0.6. Purified phage (250 μL) was added, and the culture was incubated overnight. Bacterial debris was removed by centrifugation (10,000 × g, 20 min, 4°C) and filtration through a 0.22 μm membrane. DNase I and RNase A were added to the filtrate and incubated at room temperature for 30 minutes.
The phage particles were precipitated by adding NaCl to a final concentration of 1 M, followed by PEG-8000 to 10% (w/v), with stirring. The solution was incubated on ice for at least 1 hour and then centrifuged at 11,000 × g for 20 minutes at 4°C. The pellet was resuspended in 1.6 mL SM Buffer. Residual PEG and contaminants were removed by repeated chloroform extraction (3-5 cycles) until the aqueous phase was clear. The concentrated phage titer was determined and stored at 4°C.

#### 3.2. Phage Spot Titer Assay

A fresh colony of *E. coli* MG1655 was inoculated into 5 mL LB medium and grown to the exponential phase at 37°C with shaking.
The phage lysate was serially diluted (up to 10-9) in sterile LB broth. A 10 μL aliquot of each dilution was spotted onto a lawn of the host bacterium prepared in a double-layer agar assay (bottom layer: 1.5% agar-LB; top layer: 0.7% agar-LB mixed with 1 mL of bacterial culture).
Spots were allowed to dry, and plates were incubated at 37°C for 8-12 hours. Plaques were counted to determine the phage titer in Plaque-Forming Units per mL (PFU/mL).

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{% block nav\_text\_1 %}▶ Overview{% endblock %}
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# Overview

CAU-China is committed to establishing a highly controllable, secure, and efficient precision antibacterial platform for the production of bacteriophage particles. The purpose of this page is to present the results of the design to provide experimental evidence for the validation of the ideas.

# **1. Verification of Phage-like particle Synthesis and Infectivity**

To enhance visualization during the verification of Phage-like particle synthesis and infectivity, we replaced the *MazF* toxin gene on the plasmid with *bjGFP* and conducted the following experiments.

![Fig. 1](https://static.igem.wiki/teams/5846/wiki/result/fig-1.avif)

Figure 1: The diagram of packaging visualization plasmid.

## **Plasmid construction and validation**

Using our company’ s synthetic pUC19-T7 packaging sequence plasmid as a backbone, we linearized the vector via reverse PCR and then performed seamless cloning with the PTetA and *bjGFP* containing homologous arms. The recombinant plasmid was chemically transformed into competent *Escherichia coli* MG1655 cells and plated on the LB solid medium containing ampicillin. After overnight incubation at 37°C, colonies were preliminarily screened for the recombinant plasmid via colony PCR. The correct colonies were inoculated into Ampicillin-resistant LB liquid medium under shaking condition overnight, and the plasmids were extracted and then subjected to PCR using the universal primer M13F/R (Figure 2).

Sequencing results indicate that all three plasmids were successfully constructed. T1 and T2 contained minor mutations, while T3 was error-free. T3 was used for subsequent experiments.

![Fig. 2](https://static.igem.wiki/teams/5846/wiki/result/fig-2.webp)

Figure 2: The results of plasmid PCR. The expected band size should be 2028 bp, and all three detected bands yield results consistent with expectations.
M：Star Marker D5000 Ladder; 1-3: Validation Results for the pUC19-T7 Packaging Sequence-TetA-bjGFP Construct, Designated as T1, T2, and T3

## **Verification of Correct Phage-Like Particle Synthesis**

*Escherichia coli* MG1655 containing the pUC19-packaging-PTetA-bjGFP plasmid (T3) was co-cultured with WT T7 until most bacteria lysed. Add 0.2 mL chloroform, vortex vigorously, centrifuge, and collect the supernatant. Filter through a 0.22 μm membrane to obtain T7 Mix (a mixture of phage-like particles and residual components). T7 Mix was thoroughly treated with RNase and DNase to digest any residual plasmid DNA and other impurities, yielding T7 Mix 2.0. We subjected T7 Mix 2.0 to a heat lysis reaction to release DNA from the capsid head of the phage-like particles. Universal primers M13-F/R were then used to detect specific bands via PCR.

Following PCR, agarose gel electrophoresis revealed the target specific band, and sequencing confirmed the sequence was entirely correct. Simultaneously, PCR performed on the control WT T7 lysate did not yield any relevant specific bands, ruling out background interference (Figure 3).

![Fig. 3](https://static.igem.wiki/teams/5846/wiki/result/fig-3.webp)

Figure 3: The results of PCR performed on T7 Mix 2.0 after thermal cracking.
M：Star Marker D5000 ladder; 1: The PCR products from the T7 Mix 2.0 heat-lysed samples; 2: The PCR products from the WT T7 phage heat-lysed samples

## **Verification of Phage-Like Particle Infectivity**

We cultured WT *Escherichia coli* MG1655 in liquid LB medium and added the phage inhibitor (CRGEN phage inhibitor, PR0401) at a 1:500 ratio. When the optical density reached 0.7, WT *Escherichia coli* MG1655 was mixed with T7 Mix 2.0 and cultured in liquid LB for 1.5 hours. The cells were then harvested, resuspended three times in LB to wash off T7 Mix as thoroughly as possible. Finally, the cells were spread onto LB plates containing 50 μg/mL ampicillin (Amp) and incubated at 37°C overnight.

We observed target colonies emitting green fluorescence, confirming that the phage-like particles successfully infected their host bacteria and injected the DNA concatemer into WT *Escherichia coli* MG1655. The reporter gene *bjGFP* was successfully expressed.

![Fig. 4](https://static.igem.wiki/teams/5846/wiki/result/fig-4.webp)

Figure 4: Phage inhibitor-treated *E. coli* MG1655 co-cultured with T7 Mix 2.0.

To further validate the successful expression of *bjGFP* on the DNA concatemer in the recipient bacteria, we picked fluorescent colonies from Figure 4 and cultured them overnight in ampicillin-resistant LB liquid medium as the experimental group. Concurrently, WT *Escherichia coli* MG1655 was cultured overnight in LB liquid medium as the control group.

Results showed that the overnight culture of the experimental group exhibited intense green fluorescence upon UV excitation. In contrast, the control group showed no fluorescent signal under identical conditions (Figure 5). After centrifuging both groups to collect bacterial cells, this difference remained clearly visible under natural light. The bacterial pellet from the experimental group exhibited a characteristic green color due to *bjGFP* expression, while the control group’ s pellet appeared as the typical milky white color. This phenomenon strongly demonstrates the stable and high-level expression of the *bjGFP* gene on the DNA concatemer delivered by phage-like particles within the recipient bacteria (Figure 6).

![Fig. 5](https://static.igem.wiki/teams/5846/wiki/result/fig-5.webp)

Figure 5. Visible color difference of overnight culture between the control and experimental groups.Left: overnight culture of the experimental group. Right: overnight culture of the control group.

![Fig. 6](https://static.igem.wiki/teams/5846/wiki/result/fig-6.webp)

Figure 6. Visible color difference of cell pellets between the control and experimental groups.Left: overnight culture of the experimental group. Right: overnight culture of the control group.

# **2. Functional Validation of Toxin-Antitoxin System**

Due to time constraints, we have not yet integrated the designed MazE-carrying fragment into our chassis organism genome. Therefore, we opted to construct the following toxin-antitoxin (TA) system for preliminary functional validation.

## **Construction and Validation of the Four Plasmids**

Using pUC19 as the backbone, we linearized the vector via reverse PCR. Subsequently, we obtained the araC-pBAD DNA fragment from pKD46 and the DNA fragments encoding the antitoxin gene *MazE* and toxin gene *MazF* from the WT *Escherichia coli* MG1655 genome. These were then seamlessly cloned. Subsequently, the recombinant plasmids were chemically transformed into DH5α and spread onto Amp-resistant LB solid medium. After overnight incubation at 37°C, colonies were preliminarily screened for recombinant plasmids via colony PCR. The correct colonies were inoculated into ampicillin-resistant LB liquid medium under shaking incubation overnight. Plasmids were extracted and subjected to PCR verification.

Sequencing results confirmed successful construction of all four plasmids. We selected M1, M2, M3-1, and M4-1 for subsequent functional validation of the TA system.

![Fig. 7](https://static.igem.wiki/teams/5846/wiki/result/fig-7.avif)

Figure 7: Four diagrams used to validate the toxin-antitoxin system.

![Fig. 8](https://static.igem.wiki/teams/5846/wiki/result/fig-8.webp)

Figure 8: The results of plasmid PCR. The correct band using M1 as the template should be 1553bp. The correct band using M2 as the template should be 1807bp.
M: Star Marker D5000 ladder; 1: The PCR results for M1 using jc1-F and jc2-R primers; 2: The PCR results for M2 using jc1-F and jc2-R primers

![Fig. 9](https://static.igem.wiki/teams/5846/wiki/result/fig-9.webp)

Figure 9: The results of plasmid PCR. The correct band using M3 as the template should be 1922bp. The expected band using M4 as the template should be 2185bp.
M: Star Marker D5000 ladder; 1: The PCR results for M3-1 using jc1-F and jc2-R primers; 2: The PCR results for M3-2 using jc1-F and jc2-R primers; 3: The PCR results for M4-1 using jc1-F and jc2-R primers; 4: The PCR results for M4-2 using jc1-F and jc2-R primers

### **Functional Validation of the TA system in *E. coli* MG1655**

We individually transformed the four successfully constructed plasmids into our chassis strain, *E. coli* MG1655, and tested cell toxicity on plates with or without 0.2% L-arabinose. When *MazE* or *MazE-MazF* were induced by 0.2% L-arabinose, transformant colonies were formed on the plate. However, when *MazF* was induced by 0.2% L-arabinose in the presence of 0.2% arabinose, no colonies were formed (Figure 10).

We also examined the effects of each group of transformed bacteria on inducing cell growth in liquid culture. As shown in Figure 11, growth inhibition was immediately observed in the *MazF* group following induction starting at 90 min, upon the presence of 0.2% arabinose, whereas no such phenomenon was observed in the *MazE* group or the *MazE-MazF* group. The pBAD group served as the negative control. The results indicate that *MazF*, as the toxin in the TA system, significantly inhibits cell growth, while *MazE*, as the antitoxin within the same operon, effectively neutralizes the toxicity of *MazF*.

![Fig. 10](https://static.igem.wiki/teams/5846/wiki/result/fig-10.webp)

Figure 10: Toxicity of *MazF* on plates.

![Fig. 11](https://static.igem.wiki/teams/5846/wiki/result/fig-11.webp)

Figure 11: Effect of *MazF* on cell growth induction.

### **Functional Validation of the TA system in *Pseudomonas syringae* pv. *tomato* DC3000**

We individually transformed the four successfully constructed plasmids into *Pseudomonas syringae* pv. *tomato* DC3000, and cell toxicity was tested on plates with or without 0.2% L-arabinose. When *MazE* or *MazE-MazF* were induced, transformant colonies were formed on the plate. However, when *MazF* was induced in the presence of 0.2% arabinose, no colonies were formed (Figure 12).

![Fig. 12](https://static.igem.wiki/teams/5846/wiki/result/fig-12.webp)

Figure 12: Toxicity of *MazF* on plates.

In our TA system, *MazF* effectively inhibits the growth of the chassis strain *E. coli* MG1655 and the pathogenic strain DC3000 upon induction, leading to their death. The complementary MazE antitoxin effectively antagonizes MazF toxicity, ensuring the normal survival of the chassis strain. This TA system provides crucial safety assurance for our subsequent work in safely producing phage-like particles carrying toxin genes within the chassis strain.

# **Split the T7 genome and construct an inducible expression system for controlled production of phage-like particles**

To enable *E. coli* MG1655 to autonomously produce the phage protein capsid and ensure that plasmids containing replication origins can undergo rolling-circle replication in *E. coli* MG1655 in the same manner as the T7 genome, we performed segmentally recombining the T7 genome (with gp1 removed) into the genome of *E. coli* MG1655 using pKD46-SacB (carrying the SacB gene for sucrose sensitivity screening).

## **Construction and Verification of Phage Genome Recombinant Vector**

To achieve genetic recombination, we constructed four plasmids inserted with four segments of the phage genome. Using homologous double crossover, we accomplished the integration of the T7 phage genome into the genome of MG1655.

In order to ensure the integrity of the T7 phage structural proteins during this part of the validation, we maintained the integrity of the structural protein region-Class III genes by not deleting *gp17* in our experiments.

![Fig.13](https://static.igem.wiki/teams/5846/wiki/result/fig-13.webp)

Figure 13: Four diagrams of T7 phage genome recombination

We extracted phage genomic DNA from the supernatant of the lysate using the λ Phage Genomic DNA Rapid Extraction Kit, then diluted it to a concentration of 10 ng/μL for PCR amplification of genomic fragments, and successfully obtained all target genomic fragments (Figure 13).

![Fig.14](https://static.igem.wiki/teams/5846/wiki/result/fig-14.webp)

Figure 14: The result of genome PCR. 1-1: 2910bp; 1-2: 1760bp; 2: 11438bp; 3: 9289bp; 4-1: 12421bp; 4-2: 434bp. The size of each band was correct and the sequencing results proved the correctness of the fragments.
M: Star Marker 1kb Ladder; 1: Template amplification obtained by thermal lysis 4-2; 2-7: Extraction of phage genome as template amplification of fragments T7g-1-1, T7g-1-2, T7g-2, T7g-3, T7g-4-1, T7g-4-2

To achieve the screening of homologous recombination via double crossover, we constructed the pKD46-SacB vector by inserting the promoter and coding sequence of *SacB* into the pKD46 vector.

![Fig.15](https://static.igem.wiki/teams/5846/wiki/result/fig-15.webp)

Figure 15: The result of pKD46-SacB test. The correct band should be 2282 bp, and all nine band results are correct.
M: Star Marker D5000 Ladder; 1-9: pKD46-SacB test

During the construction of the four recombinant plasmids, the homologous arms for seamless cloning carried by Down-armT7 are different. Therefore, we amplified three types of Down-armT7 with different homologous arms from the genome of *E. coli* MG1655 using PCR, and simultaneously amplified Up-armT7 with homologous arms.

We constructed the backbone of the first recombinant plasmid, pKD46-SacB-up-down, by performing seamless cloning of pKD46-SacB linearized via inverse PCR with Up-armT7 and Down-armT7-1. For the backbones of the second and third recombinant plasmids, we conducted seamless cloning of linearized pKD46-SacB with Down-armT7-2. The backbone of the fourth recombinant plasmid was constructed by seamless cloning of linearized pKD46-SacB with T7g-4-2 and Down-armT7-3. The above products were individually transformed into *E. coli* DH5α via the heat shock method, plated on LB agar plates containing Amp, and incubated at 28°C. The construction of the plasmids was verified by colony PCR.

![Fig.16](https://static.igem.wiki/teams/5846/wiki/result/fig-16.webp)

Figure 16: The result of pKD46-SacB-up-down test. The correct band should be 1168 bp, and all eight band results are correct.
M: Star Marker D5000 Ladder; 1-8: pKD46-SacB-up-down test

![Fig.17](https://static.igem.wiki/teams/5846/wiki/result/fig-17.webp)

Figure 17: The result of pKD46-SacB-down test. The correct band should be 666 bp, and all three band results are correct.
M: Star Marker D5000 Ladder; 1-3: pKD46-SacB-down test

![Fig.18](https://static.igem.wiki/teams/5846/wiki/result/fig-18.webp)

Figure 18: The result of pKD46-SacB-4-2-down test. The correct band should be 1076 bp, and all two band results are correct.
M: Star Marker D5000 Ladder; 1-2: pKD46-SacB-4-2-down test

After obtaining the required vectors, we extracted the corresponding three plasmids from *E. coli* DH5α and linearized them via inverse PCR. We constructed pKD46-SacB-1-1 by performing seamless cloning of the linearized pKD46-SacB-up-down with T7g-1-1. For the linearized pKD46-SacB-down, we conducted seamless cloning with T7g-2 and T7g-3 respectively to construct two recombinant plasmids: pKD46-SacB-2 and pKD46-SacB-3. Additionally, we constructed the recombinant plasmid pKD46-SacB-4 by seamless cloning of the linearized pKD46-SacB-4-2-down with 4-1. The above seamless cloning products were transformed into *E. coli* DH5α via the heat shock method, plated on LB agar plates containing Amp, and incubated at 28°C. The construction of the plasmids was verified by colony PCR.

![Fig.19](https://static.igem.wiki/teams/5846/wiki/result/fig-19.webp)

Figure 19: The result of pKD46-SacB-1-1 test. The correct band should be 4032 bp, and 1、2、4 band results are correct.
M: Star Marker 1kb Ladder; 1-4: pKD46-SacB-1-1 test

![Fig.20](https://static.igem.wiki/teams/5846/wiki/result/fig-20.webp)

Figure 20: The result of pKD46-SacB-2 test and pKD46-SacB-3. The correct band should be 483 bp(pKD46-SacB-2) and 503 bp(pKD46-SacB-3), and all five band results are correct.
M: Star Marker 1kb Ladder; 1-2: pKD46-SacB-2 test; 3-5: pKD46-SacB-3 test

![Fig.21](https://static.igem.wiki/teams/5846/wiki/result/fig-21.webp)

Figure 21: The result of pKD46-SacB-4 test. The correct band should be 400 bp, and 1 and 3 band results are correct.
M: Star Marker D5000 Ladder; 1-3: pKD46-SacB-4 test

After obtaining the required vectors, we extracted the corresponding three plasmids from *E. coli* DH5α. We linearized pKD46-SacB-1-1 via inverse PCR, and then performed seamless cloning of this linearized vector with T7g-1-2 to construct pKD46-SacB-1. The above seamless cloning product was transformed into *E. coli* DH5α by the heat shock method, plated on LB agar plates containing 50 μg/mL ampicillin (Amp), and incubated at 28°C. The construction of the plasmid was verified by colony PCR.

![Fig.22](https://static.igem.wiki/teams/5846/wiki/result/fig-22.webp)

Figure 22: The result of pKD46-SacB-1 test. The correct band should be 260 bp, and 1、2、3 band results are correct.
M: Star Marker D5000 Ladder; 1-5: pKD46-SacB-1 test

## **Expression Verification of Segmented Phage Genomes on the Recombinant Vector**

We extracted the plasmids (pKD46-SacB-1, pKD46-SacB-2, pKD46-SacB-3, and pKD46-SacB-4) respectively from four *E. coli* DH5α strains transformed with the recombinant plasmids. To verify whether the genomic fragments inserted into pKD46-SacB can be normally expressed, we transformed the recombinant plasmids into *E. coli* BL21 strain. With the help of T7 RNA polymerase expressed by this strain under IPTG induction, we provided conditions for the expression of the fragment genes.

We separately transformed the four plasmids into four aliquots of *E. coli* BL21 competent cells, then plated the transformed cells on Amp-containing agar plates. After screening, we inoculated the four *E. coli* BL21 strains harboring the recombinant plasmids into cultures supplemented with IPTG, followed by shaking incubation. When the bacterial culture reached an OD600 value of 1.0, the bacterial cells were collected by centrifugation. For each sample, 1 mL of bacterial cells was mixed with 80 μL of PBS and 20 μL of 5× loading buffer; after boiling water bath treatment, whole bacterial protein samples were prepared. SDS-PAGE was performed on the whole bacterial proteins (Figure 22).

![Fig.23](https://static.igem.wiki/teams/5846/wiki/result/fig-23.webp)

Figure 23: The result of bacterial whole protein SDS-PAGE Coomassie Brilliant Blue Staining.
M: Color Prestained Protein Marker; 1: WT BL21; 2: BL21 with pKD46-SacB; 3: BL21 with pKD46-SacB-1; 4: BL21 with pKD46-SacB-2; 5: BL21 with pKD46-SacB-3; 6: BL21 with pKD46-SacB-4.

From the result figure, a comparison between Lane 1 and the other lanes shows that the total bacterial proteins of *E. coli* BL21 containing the plasmid have an additional protein band near the 75 kDa position (the position indicated by the arrow in the figure 22). This indicates that after the pKD46-SacB vector entered the bacteria, it expressed proteins from the plasmid, altering the protein composition of the bacteria. When comparing Lanes 2 to 6, the genomic fragments inserted into the plasmid had no detectable effect on the bacterial proteins. There was no difference in the total protein amount of bacteria containing pKD46-SacB with inserted genomic fragments compared to those containing pKD46-SacB alone. It is inferred that the expression level of proteins encoded by the genomic fragments is very low. Since no additional bands (compared to Lane 2) were observed in Lanes 3–6, it is inferred that the expression level of proteins encoded by the genomic fragments is too low to be detected by SDS-PAGE.

Due to time constraints, we were unable to fully verify whether the gene fragments could express proteins normally. In fact, the segmentation of the genome and the expression of T7 RNA polymerase may both be factors contributing to the undetectable expression level of proteins encoded by the genomic fragments.

First, the phage genome contains numerous protein sequences with positional functions. When the genome was segmented into 4 plasmids for verifying protein expression, this segmentation might have blocked the interaction between proteins that are far apart on the genome, thereby affecting protein expression.

Second, the protein amounts of total bacterial proteins and phage proteins are not on the same order of magnitude. When the total bacterial proteins are clearly visible, the content of proteins expressed from the phage genome may be extremely low, making it impossible to distinguish and identify them.

Furthermore, since the genomic fragments were directly amplified from wild-type T7 without any optimization or modification, the coding regions are not suitable for expression under the specific expression conditions we established.

To achieve our goal, we may need to further refine and modify the phage genome, and appropriately optimize the sequences of the genomic fragments, so that they can be adapted to expression in the *E. coli* genome.

# Functional Verification of Tail Fiber-Replaced PLPs

## Tail Fiber Replacement

### 1.1 Verification of Homologous Recombination

Using the commercially synthesized pUC19-VO98\_215 plasmid as a backbone, we obtained the vector-homologous recombinant fragment via nested PCR. The pUC19 vector was linearized via reverse PCR and seamlessly cloned with the recombinant fragment. The recombinant plasmid was transformed into competent *Escherichia coli* MG1655 cells using heat shock transformation, plated on the Amp-resistant LB solid medium, and incubated overnight at 37°C. Preliminary screening for strains harboring the recombinant plasmid was performed via colony PCR and colony PCR was performed in triplicate, and all positive colonies showed the correct band size. Positive clones were picked, plasmids were extracted, and second-generation sequencing was performed. Correctly sequenced plasmids and strains harboring the correct plasmid were obtained for phage construction.

![Fig. 24](https://static.igem.wiki/teams/5846/wiki/result/fig-24.webp)

Figure 24: The results of colony PCR. Positive colonies were expected at around 2kb. All colonies in the figure matched the expectation.

![Fig. 25](https://static.igem.wiki/teams/5846/wiki/result/fig-25.webp)

Figure 25: The Result of Plasmid Sequencing

### **1.2 Verification of Successful Phage Gene Replacement**

We co-cultured the *Escherichia coli* MG1655 harboring pUC19-VO98\_215 and the *Escherichia coli* MG1655 harboring pUC19-VO98\_215(C-) with the WT T7 (wild-type T7 phage) until the majority of bacteria lysed.

![Fig. 26](https://static.igem.wiki/teams/5846/wiki/result/fig-25-n.avif)

Figure 26: The diagrams of Genome - Plasmid pUC19-VO98\_215 and pUC19-VO98\_215(C-) Homologous Recombination

After centrifugation, the supernatant was collected and filtered using a 0.22 μm membrane to obtain two T7 Mixes: T7 ∆*gp17*::*VO98\_215* and T7 ∆C-*gp17*::C-*VO98\_215*. The phage genomes were extracted from the T7 Mixes using the phage genome extraction kit (TIANGEN, DP301).

![Fig. 27](https://static.igem.wiki/teams/5846/wiki/result/fig-26-n.avif)

Figure 27: WT T7 and Isolated phage genome

Using the isolated phage genome as a template, we performed PCR with four pairs of specific primers to detect distinct bands.

![Fig. 28](https://static.igem.wiki/teams/5846/wiki/result/fig-27-n.avif)

Figure 28: Binding Status of Specific Primers.
Primer VO98\_215-up\_test showed a band only in T7 ∆*gp17*::*VO98\_215* and other primers (VO98\_215-down\_test, VO98\_215/(C-)-up, VO98\_215/(C-)-down) produced bands in all strains except wild-type T7.

Following PCR amplification, we detected the target specific band via 0.8% agarose gel electrophoresis. Sequencing results confirmed both recombinant sequences were entirely correct. Additionally, PCR reactions using the WT T7 genome as a template yielded no detectable bands, ruling out background interference.Genomic PCR was performed in triplicate. When the annealing temperature was 58°C, there was no non-specific amplification and the replicate results were consistent.

![Fig. 29](https://static.igem.wiki/teams/5846/wiki/result/fig-28-n.avif)

Figure 29: The results of phage genomic PCR.
For primers VO98\_215-up\_test and VO98\_215-down\_test, the expected positive result band size is approximately 200bp.
For primer VO98\_215/(C-)-up, the expected positive result band size is approximately 700bp.
For primer VO98\_215/(C-)-up, the expected positive result band size is approximately 1400bp.

![Fig. 30](https://static.igem.wiki/teams/5846/wiki/result/fig-30.webp)

Figure 30: Partial sequencing of recombinant genome of T7 ∆*gp17*::*VO98\_215* and T7 ∆C-*gp17*:: C-*VO98\_215* .

### 2. Verification of Successful Heterologous Expression of the Tail Protein Gene

We successfully constructed the two His-tagged plasmids: pUC19-VO98\_215-Chis and pUC19-VO98\_215(C-)-Chis. Whole-plasmid sequencing results were accurate. WT T7 infected 200 mL each of log-phase MG1655/pUC19-T7arm-VO98\_215-Chis and MG1655/pUC19-T7arm-VO98\_215(C-)-Chis cultures, which were incubated at 37°C until clarification. Filter the supernatant through a 0.22 μm membrane, add the phage precipitation reagent (20% PEG8000, 2.5 M NaCl), and precipitate overnight at 4°C. Centrifuge, dry the pellet, resuspend in 1 mL of the loading buffer, and heat at 95°C for 10 min to obtain crude protein.

Add Ni-NTA Beads to a centrifuge tube. Centrifuge, discard the supernatant, and add the Lysing Buffer. Add the sample, seal the tube, and incubate at 37°C for 1 hour. After incubation, centrifuge, aspirate and discard the supernatant. Retain the supernatant as the flow-through for electrophoresis identification. Wash with the Wash Buffer, then elute with the Elution Buffer. Incubate at room temperature for 15 minutes, then centrifuge to collect the eluate.
Use bovine serum albumin (BSA) as the standard sample and determine the protein concentration of the initial eluted sample by the Coomassie Brilliant Blue staining.
The A595 value of the protein VO98\_215-Chis is 0.246, and its concentration is calculated to be approximately 25.0μg/mL . The A595 value of the protein VO98\_215(C-)-Chis is 0.358, and its concentration is calculated to be approximately 41.2μg/mL.

![Fig. 31](https://static.igem.wiki/teams/5846/wiki/result/fig-31-nn.avif)

Figure 31: Standard Curve of Protein (left). The concentration of protein VO98\_215-Chis and VO98\_215(C-)-Chis (Right).

The remaining sample was concentrated using the ultrafiltration tube, achieving a 1000-fold concentration to obtain 30 μL of protein at a final concentration of approximately 20 μg/μL. After adding the loading buffer and mixing thoroughly, the sample was boiled at 95°C for 10 minutes to yield the purified protein sample.
The purified protein sample was used for Western blot (WB) analysis. A mouse anti-His-tag antibody (Abcam, ab18184; 1:2000 dilution in skimmed milk) served as the primary antibody, and a goat anti-mouse IgG antibody (1:2000 dilution in skimmed milk) was used as the secondary antibody. SDS-PAGE electrophoresis was performed using ACE 10% 1 mm precast gel and corresponding buffers. Adjust the loading volume to ensure 10 μg purified protein is loaded per lane. Run at 45 V until the bromophenol blue front reaches the end of the gel. Cut the gel from the rightmost three lanes for Coomassie Brilliant Blue R250 staining. Incubate at room temperature on a shaking incubator for 1 hour, then decolorize by boiling in a boiling water bath. Perform semi-dry transfer of the left gel for 12 minutes. Block with skim milk powder at room temperature on a shaking incubator for 1.5 hours. Incubate with the primary antibody (1:2000 dilution) overnight at 4°C. Wash the membrane five times with TBST for 10 minutes each. Incubate with the secondary antibody (1:2000 dilution) for 1 hour. Wash the membrane five times with TBST for 10 minutes each. Develop using the ECL chemiluminescent system with equal parts ECL Mix A and B.

![Fig. 32](https://static.igem.wiki/teams/5846/wiki/result/fig-31-n.avif)

Figure 32: Western Blot of phage WT T7, T7 ∆*gp17*::*VO98\_215* and T7 ∆C-*gp17*::C-*VO98\_215*. The expected band size of the target protein is approximately 64 kDa, and it is present only in the phage T7 ∆*gp17*::*VO98\_215* and T7 ∆C-*gp17*::C-*VO98\_215*.

## Function of Recombinant Phages with Substituted Tail Protein

Verification of phage function encompasses both adsorption and infection aspects. The double-layer plates demonstrated that WT T7 and the recombinant phages (T7 ∆*gp17*::*VO98\_215* and T7 ∆C-*gp17*::C-*VO98\_215*) failed to produce plaques directly on the *DC3000* bacterial biofilms. The phenotype could not be directly observed. Therefore, we independently validated the functionality of the recombinant phage through both adsorption and infection pathways.

![Fig. 33](https://static.igem.wiki/teams/5846/wiki/result/fig-32-n.avif)

Figure33: Double-Layer Plating

### 1. Verification of Recombinant Phage Effective Adsorption on *Pseudomonas syringae* pv. *tomato* DC3000

Wild-type T7 phage infection of MG1655/pUC19-T7arm-VO98\_215 and MG1655/pUC19-T7arm-VO98\_215(C-) yielded the recombinant phages T7 ∆*gp17*::*VO98\_215* and T7 ∆C-*gp17*::C-*VO98\_215*. After clarification, the lysate was treated with phage precipitation agents (20% PEG8000, 2.5 M NaCl), precipitated overnight at 4°C, and centrifuged. The pellet was resuspended in 1 mL SM buffer.

The resuspended sample yielded approximately 10¹¹ titer units for the titer determination.

Take 1 mL DC3000 (OD = 0.7), centrifuge at 7000xg for 5 min at 4°C, resuspend in 1 mL Ringer’ s solution. Mix 150 μL phage sample with 150 μL DC3000 and incubate for 10 min. Carbon films were floated from mica plates directly into the bacteria/phage mixtures and allowed to settle for 1 min, then stained with 1% uranyl acetate solution (prepared in deionized water) for 30 seconds. Images were acquired using a Hitachi TEM system set at an acceleration voltage of 80 kV.

![Fig. 34](https://static.igem.wiki/teams/5846/wiki/result/fig-33-n.avif)

Figure 34: Negative staining Transmission Electron Microscope detects the ability of phage with recombinant tail fiber to infect *Pseudomonas syringae* pv. *tomato* DC3000.
A: Dividing *Pseudomonas syringae* pv. *tomato* DC3000; B:*Pseudomonas syringae* pv. *tomato* DC3000; C: T7 bacteriophage; D: WT T7 can approach but cannot infect *Pseudomonas syringae* pv. *tomato* DC3000; E: T7 ∆*gp17*::*VO98\_215* infecting *Pseudomonas syringae* pv. *tomato* DC3000; F: T7 ∆C-*gp17*:: C-*VO98\_215* infecting Pseudomonas syringae pv. *tomato* DC3000.

The wild-type T7 phage (WT T7) can approach DC3000 but cannot adsorb to its surface. Furthermore, possibly due to the weak electrostatic interaction between WT T7 phage and DC3000, the number of WT T7 phages approaching a single bacterium is much lower than that of the other two recombinant phages under the same conditions.

![Fig. 35](https://static.igem.wiki/teams/5846/wiki/result/fig-35.webp)

Figure 35: WT T7 can approach but cannot infect DC3000

The recombinant phage T7 ∆*gp17*:::*VO98\_215* with full-length tail fiber replacement can approach and adsorb to the surface of DC3000. However, when compared with the infection result of phage T7 ∆C-*gp17*::C-*VO98\_215* on DC3000, it is found that the probabilities of T7 ∆*gp17*:::*VO98\_215* approaching and adsorbing to DC3000 are much lower than those of T7 ∆C-*gp17*::C-*VO98\_215*.

![Fig. 36](https://static.igem.wiki/teams/5846/wiki/result/fig-36.webp)

Figure 36: T7 ∆*gp17*:::*VO98\_215* infecting DC3000 and the enlarged partial view.

The recombinant phage T7 ∆C-*gp17*::C-*VO98\_215* with C-terminal replacement of tail fiber protein has the highest efficiency of approaching and adsorbing to the surface of DC3000. Meanwhile, we observed that almost all DC3000 adsorbed by T7 ∆C-*gp17*::C-*VO98\_215* showed cell wall damage, which implies that the possibility of T7 ∆C-*gp17*::C-*VO98\_215* infecting DC3000 is very high.

![Fig. 37](https://static.igem.wiki/teams/5846/wiki/result/fig-37.webp)

Figure 37: T7 ∆C-*gp17*::C-*VO98\_215* infecting DC3000 and the enlarged partial view. The broken cell wall is circled with the dashed lines.

When the adsorption ratios of the three phages to DC3000 were calculated under the same scale bar, significant differences were found among their adsorption ratios. This indicates that the modification of tail fibers significantly improves the adsorption capacity of phages to DC3000, and the obtained recombinant phages have strong targeting ability.

![Fig. 38](https://static.igem.wiki/teams/5846/wiki/result/fig-37-n.avif)

Figure 38: phage adsorption ratio(Left); phage number heatmap(Right).

### 2. Verification of Tail Fiber-Replaced PLPs Effective Infection of DC3000

Green fluorescence was used as the forward screening marker. The ampicillin resistance expression frame in the packaging plasmid pUC19-packaging-PTetA-GFP was replaced with a kanamycin resistance expression frame to generate pUC19-packaging-PTetA-GFP-KanR. Subsequently, the packaging plasmid pUC19-packaging-PTetA-GFP-KanR carrying the fluorescent protein bjGFP was transformed into MG1655/pUC19-VO98\_215 and MG1655/pUC19-VO98\_215(C-), to construct dual-plasmid host strains *E.coli* MG1655/pUC19-VO98\_215/pUC19-packaging-PTetA-GFP-KanR and *E.coli* MG1655/pUC19-VO98\_215(C-)/pUC19-packaging-PTetA-GFP-KanR.
Infection of the dual-plasmid host with wild-type T7 phage yielded phage-like particles(PLPs) with capsids containing only pUC19-packaging-PTetA-GFP and tail proteins replaced. These PLPs were inoculated into DC3000 liquid culture, mixed, and spread onto the resistance plates for incubation at 28°C for 16 hours. Green fluorescent colonies were observed in DC3000, indirectly confirming the infectivity of the recombinant phage against DC3000.

![Fig. 39](https://static.igem.wiki/teams/5846/wiki/result/fig-38-n.avif)

Figure 39: LB plates spread with DC3000 after co-cultivation with PLPs

The experimental results demonstrate that the PLPs successfully targeted and infected pathogens beyond the native host spectrum through tail protein replacement and packaging with the packaging plasmids.

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