iGEM 2023 — Team hubu-wuhan-china

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{% block title %}Notebook{% endblock %}
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# Notebook

# April-Knockout plasmid PK18-2.

In April, according to the instructions for the use of plasmid pK18mobsacB, we successfully integrated the third upstream and downstream fragment of ribozyme of P. chlororaphis B3-3G into plasmid pK18mobsacB, and successfully obtained knockout plasmid PK18-2.

# May-PCR and Sequencing

In May, we first used plasmid PK18-2 to knock out RNAseIII of P. chlororaphis B3-3G, and performed PCR verification and sequencing verification. Subsequently, we introduced pFLP2 plasmid and recombined kana resistance gene.

# June-KC1 plasmid expressing actin-dsRNA

In June, we constructed the KC1 plasmid expressing actin-dsRNA. In order to exclude the possible influence of non-target gene dsRNA, we also constructed KC2 plasmid expressing gfp-dsRNA.

# July-plasmids KC3 and KC4.

In July, in order to control the expression of KC1 and KC2 plasmids, we introduced LacI operator at one end of the tac promoter and transformed it into inducable dsRNA expression plasmids KC3 and KC4.

# August-KC5 and KC6 plasmids

In August, we further successfully integrated the KillerRed protein expression frame on the basis of vectors KC3 and KC4, respectively, and successfully constructed the KC5 and KC6 plasmids

# September-Biological assay and qRT-PCR

In September, we performed a biological assay and qRT-PCR to detect the expression level of actin gene in Plagiodera versicolora.

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{% block title %}Engineering Success{% endblock %}
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# Engineering

# Project Engineering

- Content
- [**1.Overview**](#item-1)
- [**2.Engineer Optimization of insect control techniques and expression systems**](#item-2)
- [**3.Engineer Optimization of insect gripping device**](#item-3)

#### **1. Overview**

Iterative design is a design method based on the cyclical process of prototyping, testing, analyzing and improving products or programs. Modifications and improvements based on the latest test results of the design iteration. The purpose of this process is to ultimately enhance the quality and functionality of the design. In the IGEM competition, we place great emphasis on engineering thinking. In the actual process of product implementation, we strive to approach the most authentic user experience. Through thorough examination of various aspects of hardware usage, we identify and address any shortcomings, in order to obtain relevant and accurate feedback. We then proceed to improve our product from different perspectives and to varying extents. Therefore, we believe that the success of the engineering aspect should be reflected in both the engineering design and the experience it provides. This year our primary focus is on optimizing the hardware.

---

#### **2.Optimization of insect control techniques and expression systems**

At the beginning of the project, we used a biocontrol bacterium called *Pseudomonas chlororaphis*B3-3G, which was selected by the guidance teacher in the laboratory and showed certain insect resistance against the blue willow leaf beetle. *Pseudomonas chlororaphis*B3-3G is sourced from the natural environment and can effectively avoid contamination from chemical pesticides. As a biocontrol bacterium, it is harmless to humans and animals. However, its insect resistance effect has a long cycle and is not very significant.

In order to enhance the insect resistance effect of *Pseudomonas chlororaphis*B3-3G, we brainstormed and consulted our teacher, and learned about the history of pesticides and the RNAi technology. RNAi technology is a gene silencing phenomenon induced by double-stranded RNA (dsRNA) in eukaryotes. Dicer, an endonuclease in the cytoplasm, cleaves dsRNA into small interfering RNAs (siRNAs) with specific lengths (21-24 nt) and structures. Then, siRNAs form RNA-induced silencing complex (RISC) with proteins such as Argonaute (AGO), which cleaves the homologous region of the corresponding gene's messenger RNA (mRNA), promoting mRNA degradation and inducing gene silencing. Our team selected the actin gene of the blue willow leaf beetle as the target gene and successfully constructed the dsRNA expression vector KC1. Additionally, we selected the gfp gene as a control and constructed the dsRNA expression vector KC2 (Figure 1).

![](https://static.igem.wiki/teams/4832/wiki/project/eng-1a.png)![](https://static.igem.wiki/teams/4832/wiki/project/eng-1b.png)

Figure 1: Carrier diagram

In addition, we are also considering other ways to enhance the insect resistance effect of *Pseudomonas chlororaphis*B3-3G. As mentioned in the design, we plan to use the dsRNA expression vector to silence the actin protein. However, *Pseudomonas chlororaphis*B3-3G possesses RNaseIII enzyme. RNaseIII is a type of endonuclease in prokaryotes that can recognize and cleave double-stranded RNA, generating small RNA molecules and participating in biological processes such as gene expression regulation and RNA degradation. To increase the accumulation of dsRNA in *Pseudomonas chlororaphis*B3-3G, we intend to knock out the RNaseIII of *Pseudomonas chlororaphis*B3-3G using the pK18mobsacB plasmid.

![](https://static.igem.wiki/teams/4832/wiki/project/eng-2.png)

Figure 2: Description of pK18mobsacB (Kvitko et al., 2011)

According to the working principle of pK18mobsacB plasmid, we successfully integrated the upstream and downstream fragments of B3-3G RNaseIII onto pK18mobsacB plasmid. In plasmid PK18-2, there is kana resistance gene, and kana resistance gene can enable host cells to survive in kana resistance environment. (Figure 3)

![](https://static.igem.wiki/teams/4832/wiki/project/eng-3.png)

Figure 3: Plasmid PK18-2

In the agar plate, we randomly selected 6 single colonies for validation. We designed 2 pairs of primers for dual-end detection to confirm the occurrence of double exchange. To ensure the accuracy of the validation, we sent the PCR products to a biological company for sequencing verification. (Figure 4-a shows the left-end PCR validation, 4-b shows the right-end PCR validation, and 4-c illustrates the sequencing results. M represents the marker, ck represents the wild-type *Pseudomonas chlororaphis*B3-3G, and 1, 2, 3, 4, 5, 6 represent the 6 selected single colonies used as templates for PCR.)

![](https://static.igem.wiki/teams/4832/wiki/project/eng-4ab.png)

Figure 4-a      Figure 4-b

![](https://static.igem.wiki/teams/4832/wiki/project/eng-4c.png)

Figure 4-c

After replacing the RNaseIII trigene of B3-3G with the resistance gene of PK18-2 plasmid. We also need to reprogram the resistance genes that are integrated into the B3-3G genome. For this purpose, we introduced a recombinant pFLP2 plasmid into B3-3G. (Figure 5)

![](https://static.igem.wiki/teams/4832/wiki/project/eng-5.png)

Figure 5: pFLP2 plasmid

After PCR detection, we can obviously see that the mutant with the kana fragment reassembled has a significantly shorter fragment length. (Figure 6-a shows PCR verification, and 6-b shows sequencing results. M was the Marker, H2O was the negative control, WT was the wild type of *Pseudomonas chlororaphis* B3-3G, CK was the above mutant that had not been transferred into pFLP plasmid, and 1,2,3,4 was the selected four single colonies as the template for PCR.)

![](https://static.igem.wiki/teams/4832/wiki/project/eng-6a.png)

Figure 6-a

![](https://static.igem.wiki/teams/4832/wiki/project/eng-6b.png)

Figure 6-b

We also sent the PCR product to a biotech company for sequencing, which confirmed that we successfully knocked out the RNase III gene and recombined the kana fragment. We successfully obtained the B3-3G mutant- KC-P, which lacks the RNase III gene.

We introduced the KC1 and KC2 plasmids into B3-3G and KC-P, respectively, and then performed northern blot verification. (Figure 7, from left to right, is Marker, KC-P+KC1, KC-P+KC2, B3-3G, B3-3G+KC1, B3-3G+KC2)

![](https://static.igem.wiki/teams/4832/wiki/project/eng-7.png)

Figure 7: northern blot results

According to our analysis of the northern blot experiment, we can clearly see a significant increase in the accumulation of the target dsRNA after knocking out RNase III.

To ensure the biosafety of our engineered bacteria after knocking out the RNase III enzyme in *Pseudomonas chlororaphis*, we introduced the lacI operator into the KC1 and KC2 strains, successfully constructing KC3 and KC4 strains to regulate dsRNA expression. Furthermore, we further integrated the KillerRed protein expression cassette into the KC3 and KC4 vectors, successfully constructing KC5 and KC6 vectors, which further ensured the safety [Read about it](https://2023.igem.wiki/hubu-wuhan-china/safety) of our engineered bacteria.（ Figure 8）

![](https://static.igem.wiki/teams/4832/wiki/project/eng-8.png)

Figure 8: Plasmid diagram

We conducted feeding experiments on detached leaves to determine the mortality rate, body weight, pupation rate, and eclosion rate of *Plagiodera versicolora* under different conditions. The experimental groups included: *Pseudomonas chlororaphis* B3-3G, *Pseudomonas chlororaphis* B3-3G+KC5, *Pseudomonas chlororaphis* B3-3G+KC6, *Pseudomonas chlororaphis* KC-P+KC5, and *Pseudomonas chlororaphis* KC-P+KC6.(Figure 9)

![](https://static.igem.wiki/teams/4832/wiki/project/resu-17.png)

Figure 9: Biological testing

Figure 10: Data analysis of the mortality rate, average weight, pupation rate, and eclosion rate of *Plagiodera versicolora*. After conducting statistical analysis, we concluded that the experimental group with *Pseudomonas chlororaphis* B3-3G showed some impact on the mortality rate, average weight, pupation rate, and eclosion rate of *Plagiodera versicolora* compared to the control group, but it did not show significant differences in statistical analysis.

After combining RNAi, the experimental group with *Pseudomonas chlororaphis* B3-3G+KC5 showed significant differences in the mortality rate, average weight, pupation rate, and eclosion rate of *Plagiodera versicolora* compared to the control group. The pupation rate decreased by approximately 46% compared to the control group, the average weight decreased by over 35% compared to the control group, and the eclosion rate decreased by approximately 48% compared to the control group.

Furthermore, we observed that the *Pseudomonas chlororaphis* KC-P+KC5, which knocked out RNase III, performed better in controlling *Plagiodera versicolora*. The experimental group with *Pseudomonas chlororaphis* KC-P+KC5 showed extremely significant differences in the mortality rate, average weight, pupation rate, and eclosion rate of *Plagiodera versicolora* compared to the control group. The pupation rate decreased by approximately 75% compared to the control group, the average weight decreased by over 60% compared to the control group, and the eclosion rate decreased by approximately 67% compared to the control group.

As shown in Figure 10-d, in the statistical analysis of the mortality rate of *Plagiodera versicolora* with *Pseudomonas chlororaphis* B3-3G+KC5, the mortality rate reached over 50% on the 5th day, while in the KC-P+KC5 experimental group after knocking out RNase III, the mortality rate reached over 50% on the 2nd day. We can see that after knocking out RNase III, the combination of *Pseudomonas chlororaphis* and RNAi greatly improved the control effect on *Plagiodera versicolora*.

![](https://static.igem.wiki/teams/4832/wiki/project/eng-10a.png)![](https://static.igem.wiki/teams/4832/wiki/project/eng-10b.png)

Figure 10-a           Figure 10-b

![](https://static.igem.wiki/teams/4832/wiki/project/eng-10c.png)![](https://static.igem.wiki/teams/4832/wiki/project/eng-10d.png)

Figure 10-c           Figure 10-d

To further validate the results, we conducted qRT-PCR analysis to detect the expression level of the actin gene in *Plagiodera versicolora* on the third day of feeding.

As shown in Figure 11, in the experimental group fed with *Pseudomonas chlororaphis* B3-3G+KC5, the expression level of the actin gene in *Plagiodera versicolora* showed significant differences compared to the control group. In the experimental group fed with *Pseudomonas chlororaphis* KC-P+KC5, the expression level of the actin gene in *Plagiodera versicolora* showed extremely significant differences compared to the control group.

By incorporating RNAi technology and optimizing the expression of dsRNA through the knockout of RNase III in *Pseudomonas chlororaphis*, we have successfully developed the KC-P+kc5 strain with significantly improved insect resistance. With these optimizations, our team has completed the optimization of insect control techniques and expression systems.

![](https://static.igem.wiki/teams/4832/wiki/project/eng-11.png)

Figure11 The expression level of actin gene

---

#### **3.Engineer Optimization of insect gripping device**

As an important component of the entire experimental process, it is necessary for us to feed the *Plagiodera versicolora* and the  *Pseudomonas chlororaphis* B3-3G obtained in the later stages test. The action of cleaning and gripping the insects is inevitable and can be time-consuming. Therefore, it has become essential to facilitate, expedite, and humanize the process of insect brush, not only for our own convenience but also for other research teams in need of insect gripping devices. We have conducted a series of market research and gathered a significant amount of information. Based on this foundation, we have designed various solutions and systematically carried out theoretical validations and implementations.

For the use of the insect brush, our first-generation product aims to fulfill the basic requirements of having sufficient support and being handheld. It should also be capable of effectively scraping both the  *Plagiodera versicolora* larvae and adults off the leaves. Therefore, when designing this solution, we took inspiration from commonly used brushes in art studios, as shown in Figure 12. However, during the later stages of usage, we discovered that this design could cause harm to the  *Plagiodera versicolora* due to excessive contact pressure from the bristles or accidental contact with the wooden handle. This is an outcome that we do not desire. Hence, we need a device that either does not directly touch the  *Plagiodera versicolora* or minimizes the impact force caused by contact.

![](https://static.igem.wiki/teams/4832/wiki/project/eng-12a.png)![](https://static.igem.wiki/teams/4832/wiki/project/eng-12b.png)

Figure 12: Design draft and real product of the first-generation wooden stick insect brush

Therefore, in the second generation product, we replaced the fixed bristle wooden stick with a device similar to a disposable pipette in the laboratory, attempting to blow the insects down with gas. As shown in Figure 13A, this device not only avoids harming the insects but also retains the assistance of the bristles from the first generation for insect brush.

![](https://static.igem.wiki/teams/4832/wiki/project/eng-13a.png)

Figure 13A: insect brush with suction capsule (design drawing)

![](https://static.igem.wiki/teams/4832/wiki/project/eng-13b.png)

Figure 13B: replaceable brush heads of different sizes

After discussing, we have decided to add an illumination end to the front of theinsect gripping device, which will provide a more intuitive observation of the morphological changes and characteristics of the insects during collection. It will be clearer and more convenient than observing directly under a fluorescent lamp. A lightweight and compact LED is undoubtedly the most suitable tool for this purpose. However, during the actual production process, we found that it was difficult to install the LED.After weighing the options, we decided that if the LED was installed directly at the mouth of the second-generation insect collector, it would block the original outlet and render the jet function ineffective. If the light tube was wrapped around the upper end of the device, it would also violate our original intention of requiring convenience for the device. Therefore, we decided to extend the application scenario of the second-generation device and no longer require the illumination and air outlet functions to coexist. We changed the upper end of the second-generation insect gripping device to a push-button pen tube and assembled the button battery and LED inside the pen tube to make a small flashlight with bristles at the front, as shown in Figure 13A. When using the device, the operator only needs to press the device like a pen to use it, as shown in Figure 13B. This device is low-cost, lightweight, compact and reusable. This solution is not only suitable for the collection of  *Plagiodera versicolora* in our laboratory, but also a good tool for observing other organisms. This is also a major extension function of the third-generation insect brush.

![](https://static.igem.wiki/teams/4832/wiki/project/eng-14a.png)

Figure 14A: picture of real product

![](https://static.igem.wiki/teams/4832/wiki/project/eng-14b.png)

Figure 14B:picture of actual operation

Finally, after three generations of updates and iterations, we have designed the final two generations of products. In order to improve the practicality and user-friendliness of the product, our complete set of products will be accompanied by disposable gloves, the culture boxes for the *Plagiodera versicolora* , tweezers, and other equipment for use. Considering that our insect gripping device toolkit may be used by people of different ages, educational backgrounds and research directions, we have also taken photos of the various versions of the toolkit in use to facilitate users' correct operation.

![](https://static.igem.wiki/teams/4832/wiki/project/eng-15.png)

Figure 15:picture of actual operation

---

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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# Procotol

# Project Protocol

The techniques we used in our project are as the pdf follows

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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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# Results

# Project Results

- Content
- [**1.Overview**](#item-1)
- [**2.Entire experimental project**](#item-2)
- [**3.Knockout of RNase III Gene**](#item-3)
- [**4.Expression Vectors**](#item-4)
- [**5.B3-3G and KC-P**](#item-5)
- [**6.Safety Switch**](#item-6)
- [**7.*Plagiodera versicolora*'s bioassay**](#item-7)
- [**8.**qRT-PCR detects](#item-8)

#### **1.Overview**

In our project, after consulting with relevant instructors and conducting a series of preliminary investigations, we decided to utilize a biocontrol bacterium, *Pseudomonas chlororaphis*  B3-3G, identified by the mentor's research group for its insect-resistant properties against the *Plagiodera versicolora*. We integrated this bacterium with RNAi technology to achieve a safe and effective control of the *Plagiodera versicolora*.

---

#### **2.The entire experimental project is divided into four modules**

1. Knockout of RNase III: RNase III, a key enzyme for dsRNA degradation in prokaryotes, was targeted for knockout to enhance the accumulation of dsRNA expression in *Pseudomonas chlororaphis* B3-3G. Using the pK18mobsacB plasmid, we successfully knocked out RNase III, resulting in the mutant strain KC-P.

2. Construction of dsRNA Expression Vectors and Expression in *Pseudomonas chlororaphis* B3-3G and KC-P: The actin gene of the *Plagiodera versicolora*was selected as the target gene, and we successfully constructed the dsRNA expression vector KC1. Simultaneously, the gfp gene was chosen as a control, and the dsRNA expression vector KC2 was constructed. Subsequently, KC1 and KC2 were separately transformed into the wild-type *Pseudomonas chlororaphis* B3-3G and the mutant strain KC-P, achieving dsRNA expression. Furthermore, Northern blot analysis revealed higher expression of actin dsRNA in the mutant strain KC-P, contributing to increased accumulation in *Pseudomonas chlororaphis* B3-3G.

3. Construction and Validation of a Safety Switch: To ensure the biosafety of our engineered bacteria, LacI operators were introduced into the KC1 and KC2 vectors, leading to the successful construction of KC3 and KC4 for regulating dsRNA expression. Additionally, KillerRed protein expression cassettes were further integrated into the KC3 and KC4 vectors, resulting in the construction of KC5 and KC6 vectors to enhance the safety of the engineered bacteria.

4. Validation of *Pseudomonas chlororaphis* B3-3G Combined with RNAi for *Plagiodera versicolora*Control: Through feeding experiments on detached leaves, we measured relevant data such as mortality, weight, pupation rate, and eclosion rate of the *Plagiodera versicolora*in the experimental and control groups. At the molecular level, qRT-PCR was utilized to detect the expression levels of the actin gene. The results demonstrated that the combination of *Pseudomonas chlororaphis* B3-3G and RNAi improved the control effectiveness against the *Plagiodera versicolora*.

---

#### **3. Knockout of RNase III Gene**

In prokaryotes, RNase III is the primary enzyme for dsRNA degradation. After discussions with our principal investigator (PI), we decided to use the pK18mobsacB plasmid to knockout the RNase III gene.

![](https://static.igem.wiki/teams/4832/wiki/project/resu-1.png)

Figure 1 How pK18mobsacB works (Kvitko et al., 2011)

According to the working principle of the pK18mobsacB plasmid (as illustrated in Figure 1), we successfully integrated the upstream and downstream fragments of RNase III from *Pseudomonas chlororaphis* B3-3G into the pK18mobsacB plasmid, resulting in the knockout plasmid PK18-2.

![](https://static.igem.wiki/teams/4832/wiki/project/resu-2.png)

Figure2

(PK18-2 plasmid, where "Down sequence" represents the downstream fragment of RNase III in *Pseudomonas chlororaphis* B3-3G, and "Up sequence" represents the upstream fragment of RNase III.)

Within the PK18-2 plasmid, there are kana resistance genes and SacB genes. Through electroporation, we introduced this plasmid into B3-3G, aiming to achieve the knockout of the RNase III gene. The SacB gene expresses levansucrase, catalyzing the hydrolysis of sucrose into levans that are lethal to cells. Simultaneously, the kana resistance gene allows host cells to survive in a kanamycin-resistant environment.

After electroporation, a period of antibiotic-free cultivation was initiated to recover transformed bacteria. Subsequently, the bacteria were spread on plates containing 15% sucrose and 100 mg/mL kanamycin to screen for mutants. The results were as follows:

|  | Kana Resistance | Survival in Sucrose Environment | Theoretical Result |
| --- | --- | --- | --- |
| B3-3G (No homologous recombination) | No | Yes | Death |
| B3-3G Single exchange (Plasmid integrated into the genome) | No | Yes | Death |
| B3-3G (Homologous recombination, double exchange) | No | Yes | Survival |

On the sucrose-kanamycin plates, six randomly selected single colonies were verified through PCR. Primers were designed with one primer matching the original genomic sequence and the other matching the sequence on the integrated plasmid. PCR testing showed that colonies 1 and 6 exhibited the expected results, confirming them as mutants with double exchange events. To further validate these findings, the PCR products were sequenced, confirming that colonies 1 and 6 were indeed the desired mutants (as shown in Figure 3-a, 3-b, and 3-c).

![](https://static.igem.wiki/teams/4832/wiki/project/resu-3.png)

Figure 3-a shows left-end PCR verification, 3-b is right-end PCR verification, and 3-c is a schematic diagram of sequencing results. M is Marker, ck is *Pseudomonas chlororaphis* B3-3G wild-type, 1, 2, 3, 4, 5, 6, PCR is performed using the selected 6 single colonies as a template.

With the replacement of the RNase III gene in B3-3G by the resistance gene in the PK18-2 plasmid, the next step involved the removal of the resistance gene integrated into the B3-3G genome.

![](https://static.igem.wiki/teams/4832/wiki/project/resu-4.png)

Figure 4 - pFLP2 plasmid

Through large-scale dot-blotting, we selected colonies on plates with 100 mg/mL kanamycin and 500 mg/mL ampicillin. The high concentration of ampicillin was used because B3-3G inherently possessed some resistance to ampicillin, and after testing, it was determined that a higher concentration was necessary to ensure the death of B3-3G not transformed with the pFLP2 plasmid.

**Results from the selection process on kanamycin and ampicillin plates were as follows:**

|  | Kana Resistance (100 mg/mL) | Amp Resistance (500 mg/mL) | Theoretical Result |
| --- | --- | --- | --- |
| B3-3G (No homologous recombination) | Yes | No | Survival on kana plate, death on amp plate |
| B3-3G + pFLP2 plasmid | Yes | Yes | Survival on both kana and amp plates |
| B3-3G (Homologous recombination) | No | No | Death on both kana and amp plates |
| B3-3G + pFLP2 plasmid | No | Yes | Death on kana plate, survival on amp plate |

According to the results in Table 1, colonies that survived on the ampicillin plate but died on the kanamycin plate were selected for further verification.

![](https://static.igem.wiki/teams/4832/wiki/project/resu-5.png)

Figure 5 - Results of dot-blotting on kanamycin and ampicillin plates

Primers were designed with one primer on the Down sequence and the other on the Up sequence. PCR testing revealed that *Pseudomonas chlororaphis* B3-3G amplified a 2401 bp DNA fragment, non-homologous recombination resulted in a 3226 bp DNA fragment, and homologous recombination produced an 1877 bp DNA fragment. Results from the PCR verification indicated that all four selected colonies exhibited the expected results, confirming that they had undergone homologous recombination. Further sequencing confirmed the successful homologous recombination events.

![](https://static.igem.wiki/teams/4832/wiki/project/resu-6.png)

Figure 6-a, 6-b ,PCR verification and sequencing results

In conclusion, we successfully obtained the mutant strain of *Pseudomonas chlororaphis* B3-3G, named KC-P, by knocking out the RNase III gene.

---

#### **4. Construction of dsRNA Expression Vectors**

We selected the actin gene of the *Plagiodera versicolora*as our target gene and proceeded to construct dsRNA expression vectors based on this gene.

As illustrated in Figure 7, we incorporated tac promoters into both segments of the actin gene sequence, leading to the construction of the KC1 plasmid for expressing actin-dsRNA. To mitigate the potential impact of non-target gene dsRNA, we also constructed the KC2 plasmid for expressing gfp-dsRNA.

![](https://static.igem.wiki/teams/4832/wiki/project/resu-7.png)

![](https://static.igem.wiki/teams/4832/wiki/project/resu-8.png)

Figure 7-KC1 and KC2 plasmid plots

---

#### **5. Expression of dsRNA in *Pseudomonas chlororaphis* B3-3G and *Pseudomonas chlororaphis* KC-P**

By electroporation, we introduced KC1 and KC2 plasmids into *Pseudomonas chlororaphis* B3-3G and *Pseudomonas chlororaphis* KC-P, respectively, and then used the actin sequence as a probe for northern blot verification, knot
The result is shown in Figure 8.

![](https://static.igem.wiki/teams/4832/wiki/project/resu-9.png)

Figure 8 presents the northern blot detection results from left to right, including Marker, KC-P+KC1, KC-P+KC2, B3-3G, B3-3G+KC1, and B3-3G+KC2.

According to Figure 8, it is evident that the accumulation of actin-dsRNA significantly increased after the knockout of RNase III.

---

#### **6. Construction and Verification of the Safety Switch**

**1.Construction of the LacI Operator Switch**

Considering that the dsRNA expression in KC1 and KC2 plasmids is constitutive, we introduced a LacI operator at one end of the tac promoter, transforming it into an inducible dsRNA expression plasmid for both KC3 and KC4. The expression of dsRNA was regulated by IPTG.

![](https://static.igem.wiki/teams/4832/wiki/project/resu-10.png)

![](https://static.igem.wiki/teams/4832/wiki/project/resu-11.png)

Figure 9 illustrates the KC3 and KC4 plasmids.

Through electroporation, we introduced KC3 and KC4 plasmids into the *Pseudomonas chlororaphis* KC-P. Subsequently, using the actin sequence as a probe, we conducted northern blot verification, and the results are shown in Figure 10.

![](https://static.igem.wiki/teams/4832/wiki/project/resu-12.png)

Figure 10 represents the northern blot results from left to right, including Marker, KC-P+KC3+IPTG, KC-P+KC4+IPTG, KC-P+KC3, KC-P+KC3, and KC-P+KC4.

According to Figure 10, the LacI operator significantly inhibits the expression of actin-dsRNA. However, the KC-P+KC3 without IPTG induction still exhibits slight coloration. We believe there are two possible reasons for this result:

The LacI operator is not a completely stringent switch, allowing KC-P to express a small amount of actin-dsRNA.

We added the LacI operator only between one tac promoter and the actin sequence, while the other tac promoter can still transcribe actin-ssRNA in the absence of IPTG induction. Although we introduced RNAase in the Northern blot experiment to degrade actin-ssRNA, there may still be a small amount of actin-ssRNA binding to the probe.

Based on the analysis of Figure 10, we can still observe a certain level of inhibition in the expression of actin-dsRNA without induction.

**2. The Second Switch: KillerRed**

Our team places particular emphasis on biosafety. After introducing the LacI operator, we decided to add a suicide switch—KillerRed. The KillerRed protein, expressed by the killerRed gene, undergoes a structural change upon light stimulation, generating highly toxic oxygen radicals in cells, leading to cell death.

To achieve this, we further integrated the KillerRed protein expression cassette into the KC3 and KC4 plasmids, successfully constructing the KC5 and KC6 plasmids (Figure 11), further ensuring the safety of our engineered bacteria.

![](https://static.igem.wiki/teams/4832/wiki/project/resu-13.png)

![](https://static.igem.wiki/teams/4832/wiki/project/resu-14.png)

Figure 11 illustrates the KC5 and KC6 plasmids.

Through electroporation, we introduced the KC5 and KC6 plasmids into the *Pseudomonas chlororaphis* KC-P and validated them.

The results in Figure 12 indicate that on the plate after 1 hour of light exposure, the number of viable bacteria has already decreased by more than 95%, demonstrating the excellent lethality of our plasmids.

![](https://static.igem.wiki/teams/4832/wiki/project/resu-15.png)

Figure 12, from left to right, represents plates after 0h, 0.5h, 1h, and 2h of light exposure.

We also conducted modeling analysis using ordinary differential equations to describe the system, where:

d[N]dt=−k[N]

N represents the number of viable bacteria,t represents the light exposure time,k represents the bacterial death rate

We used the ode45 function in MATLAB to solve this differential equation, compared the results with actual data, and calculated the error. Then, we used the fminsearch function to fit the unknown parameter k, minimizing the error. Since there might be multiple local optimal solutions, we performed multiple fittings to find the global optimal solution. Based on the fitting results, we re-solved the differential equation to obtain the final model and plotted the fitting curve, as shown in the figure. The fitted value for the parameter k in the exponential fit was 0.07.

**Exponential Fit:**

Using the MATLAB Curve Fitting Toolbox to fit the existing data, we found that an exponential fit provided the best results. The adjusted R2 was as high as 97.03%, and the root mean square error (RMSE) was 5.883. The final expression for the fitted curve is:

f(x)=98.75∗exp∧⁡((−0.06922)∗x)

![](https://static.igem.wiki/teams/4832/wiki/project/resu-16.png)

Figure: represents our data analysis modeling for the KillerRed suicide switch.

This model is similar to the logarithmic residual model of high-temperature sterilization bacteria. From the experimental results and model predictions, it can be observed that after introducing KillerRed, the majority of bacteria can be killed with only a short period of light exposure. However, unlike high-temperature sterilization, it does not completely eliminate almost all bacteria. This also indicates that compared to physical methods of killing, the bacterial death caused by the biological suicide switch is relatively gentle, yet sufficient to meet our biosecurity needs.

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#### **7. *Pseudomonas chlororaphis* combined with RNAi to resist the *Plagiodera versicolora's* bioassay**

We conducted feeding experiments on detached leaves to determine the mortality rate, body weight, pupation rate, and eclosion rate of *Plagiodera versicolora*under different conditions. The experimental groups included: *Pseudomonas chlororaphis* B3-3G, *Pseudomonas chlororaphis* B3-3G+KC5, *Pseudomonas chlororaphis* B3-3G+KC6, *Pseudomonas chlororaphis* KC-P+KC5, and *Pseudomonas chlororaphis* KC-P+KC6.(Figure 9)

![](https://static.igem.wiki/teams/4832/wiki/project/resu-17.png)

Figure 13: Data analysis of the mortality rate, average weight, pupation rate, and eclosion rate of *Plagiodera versicolora*.

After conducting statistical analysis, we concluded that the experimental group with *Pseudomonas chlororaphis* B3-3G showed some impact on the mortality rate, average weight, pupation rate, and eclosion rate of *Plagiodera versicolora*compared to the control group, but it did not show significant differences in statistical analysis.

![](https://static.igem.wiki/teams/4832/wiki/project/resu-18.png)

After combining RNAi, the experimental group with *Pseudomonas chlororaphis* B3-3G+KC5 showed significant differences in the mortality rate, average weight, pupation rate, and eclosion rate of *Plagiodera versicolora*compared to the control group. The pupation rate decreased by approximately 46% compared to the control group, the average weight decreased by over 35% compared to the control group, and the eclosion rate decreased by approximately 48% compared to the control group.

Furthermore, we observed that the *Pseudomonas chlororaphis* KC-P+KC5, which knocked out RNase III, performed better in controlling *Plagiodera versicolora*. The experimental group with *Pseudomonas chlororaphis* KC-P+KC5 showed extremely significant differences in the mortality rate, average weight, pupation rate, and eclosion rate of *Plagiodera versicolora*compared to the control group. The pupation rate decreased by approximately 75% compared to the control group, the average weight decreased by over 60% compared to the control group, and the eclosion rate decreased by approximately 67% compared to the control group.

As shown in Figure 14-d, in the statistical analysis of the mortality rate of *Plagiodera versicolora*with *Pseudomonas chlororaphis* B3-3G+KC5, the mortality rate reached over 50% on the 5th day, while in the KC-P+KC5 experimental group after knocking out RNase III, the mortality rate reached over 50% on the 2nd day.

We can see that after knocking out RNase III, the combination of *Pseudomonas chlororaphis* and RNAi greatly improved the control effect on Plagiodera versicolora.

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#### **8. QRT-PCR detects the expression level of the actin gene in Plagiodera versicolora**

To further validate the results, we conducted qRT-PCR analysis to detect the expression level of the actin gene in *Plagiodera versicolora*on the third day of feeding.

![](https://static.igem.wiki/teams/4832/wiki/resu-o1.png)

FIG 15: The expression level of actin gene*Plagiodera versicolora*.

As shown in Figure 15, in the experimental group fed with *Pseudomonas chlororaphis* B3-3G+KC5, the expression level of the actin gene in *Plagiodera versicolora*showed significant differences compared to the control group. In the experimental group fed with *Pseudomonas chlororaphis* KC-P+KC5, the expression level of the actin gene in *Plagiodera versicolora*showed extremely significant differences compared to the control group.

By incorporating RNAi technology and optimizing the expression of dsRNA through the knockout of RNase III in *Pseudomonas chlororaphis*, we have successfully developed the KC-P+kc5 strain with significantly improved insect resistance. With these optimizations, our team has completed the optimization of *Plagiodera versicolora*control techniques and expression systems.

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