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.
At the beginning of the project, we used a biocontrol bacterium called Pseudomonas chlororaphisB3-3G, which was selected by the guidance teacher in the laboratory and showed certain insect resistance against the blue willow leaf beetle. Pseudomonas chlororaphisB3-3G 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 chlororaphisB3-3G, 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).
Figure 1: Carrier diagram
In addition, we are also considering other ways to enhance the insect resistance effect of Pseudomonas chlororaphisB3-3G. As mentioned in the design, we plan to use the dsRNA expression vector to silence the actin protein. However, Pseudomonas chlororaphisB3-3G 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 chlororaphisB3-3G, we intend to knock out the RNaseIII of Pseudomonas chlororaphisB3-3G using the pK18mobsacB plasmid.
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)
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 chlororaphisB3-3G, and 1, 2, 3, 4, 5, 6 represent the 6 selected single colonies used as templates for PCR.)
Figure 4-a Figure 4-b
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)
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.)
Figure 6-a
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)
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 of our engineered bacteria.（ Figure 8）
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)
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.
Figure 10-a Figure 10-b
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.
Figure11 The expression level of actin gene
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.
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.
Figure 13A: insect brush with suction capsule (design drawing)
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.
Figure 14A: picture of real product
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.
Figure 15:picture of actual operation