In order to sense the up-regulation of salicylic acid signal in plants due to disease, we designed an salicylic acid biosensor as a switch in the circuit. In our design, the salicylic acid biosensor was inactivated or activated at a low dose under the background salicylic acid condition, and the maximum activation efficiency was reached after the up-regulation of salicylic acid signal in plants.

To validate the function of NahR and sal promoter ,a salicylic acid biosensor was constructed using amCyan as the reporter a constructed into plasmid pUC57.

We transformed the recombinant plasmid into E. coli BL21 (DE3) for subsequent functional verification. Before induction with salicylic acid, we verified that NahR could be expressed normally in E. coli BL21(DE3) using using 10% SDS-PAGE electrophoresis.

After the expression of NahR was verified, in order to verify the function of NahR and sal promoter, we set up a series of salicylic acid concentration gradients for induction (0 μM,0.1 μM,1 μM,10 μM,10 μM,1000 μM). E. coli BL21(DE3) strain without sal promoter was used as a blank control to explore the leakage expression of sal promoter.

We found that the expression of fluorescent proteins increased rapidly when SA concentration was between 1-10 μM. We added 200 μL of the induced culture medium inoculated with engineered bacteria to a 96-well plate, and detected the emission light intensity at 486nm using a microplate reader. Five groups of parallel replicates were set for each induced concentration.

From the above two pictures, it can be seen that sal promoter activity is extremely dependent on salicylic acid concentration, and at the same time sal has low background expression in the absence of salicylic acid.

We chose Virus-like particles (VLPs) composed of coat proteins of bacteriophage MS2 (MS2 CP) of Escherichia coli as the platform for executive functions, and displayed insecticidal proteins and plant immune activation proteins on its surface to complete the role of killing nematodes that damage plants and activating plant immunity. In order to display our functional proteins on the surface of VLP particles, we selected the SpyTag-SpyCatcher system as a scaffold for displaying functional proteins on the surface of VLP through literature review. At the same time, we also included a dual tandem 19bp stem-loop sequence that can be specifically bound by the MS2 coat protein to verify its loading capacity as a future RNA delivery vector. We plan to make MS2 coat protein express in the engineered bacteria and form VLP, bind to the functional protein with SpyCatcher on its surface, and enclose the RNA containing a 19-bp sequence of stem rings to form a multifunctional delivery.
Figure 5. Virus-like particles delivery system's function. The protein displayed on the VLP surface relies on the SpyTag-SpyCatcher covalent link, with the red portion representing RNA that can be inpacked
Our designed coat protein was constitutively expressed in the engineered bacteria. To obtain sufficient amounts of VLP, we chose to fuse the MS2 CP coding sequence to a high-copy PUC57 mini plasmid. Meanwhile, SpyCatcher-EGFP coding sequence and a 19-bp tandem stem-loop sequence were ligated downstream of the MS2 CP coding sequence (Figure 6.) . Upstream of spycatcher, we have reserved a place to integrate P_sal. Upstream of ms2 cp, we have reserved a place for access to the salicylic acid response element NahR.
Figure 6. VLP assembly plasmid vectors.
We plan to verify the connection function of induced SpyCatcher and the function of loaded RNA after the successful verification of salicylic acid regulatory elements. After homologous recombination, we obtained the target plasmid vector.
Figure 7. Colony PCR and sequencing confirmed the successful assembly.
We preserved E.coli BL21 (DE3) strains that had previously verified the correct sequence introduction. We first explored the effect of different time periods of low temperature expression on the concentration of the same volume of purified 6x His tag protein. We cultured the same 25 mL LB medium at different temperatures. The cultivation conditions are as follows.
1). LB medium containing 60 μg·mL^-1 ampicillin was added with 100 μL of preserved bacterial solution, and cultured in a shaking bed at 37℃ at 200 rpm until OD₆₀₀ was between 0.6-0.8.
2). The culture with OD₆₀₀ meeting the requirement was removed and transferred to a shaking bed at 16℃ and 160 rpm for low temperature expression for 12 h and 16 h, respectively.
3). The low-temperature culture products were placed at 4℃ and centrifuged at 6000 rpm for 30 minutes to collect bacteria.
4). The collected bacteria were re-suspended with 8 mL of PBS buffer, broken for 10 s and pause for 10 s using an ultrasonic cell crusher, and run for 30 min. Centrifuge the crushing liquid at 12000 rpm at 4℃ for 30 min, separate the supernatant and precipitate, and store them at 4℃ respectively.
5). All the supernatants were purified with Ni-NTA purification kit, and the concentration of purified samples with different expression time was determined, and SDS-PAGE experiment was performed.

The expected molecular weight of SpyCather-EGFP containing 6x His tag and the complex based on SpyCatcher-SpyTag connection were 47.4 kDa and 76.5 kDa, respectively. The result is shown in Figure 8.
Figure 8. Experimental results of protein extraction.
A. SDS-PAGE of total protein sample after 12 h expression. Line 1: Supernatant. Line 2: Precipitate. Line 3: BL21 (DE3). Line 4: Engineered bacteria introduced into the plasmid.
B. Purification results of the products expressed at 12 h. Line 1: Supernatant. Line 2: BL21 (DE3) Line 3: Engineered bacteria introduced into the plasmid. Line 4 and 6: Purified sample at 12 h. Line 5: Precipitate. Red boxes indicate bands that may be target proteins.
C. Purification results of the products expressed at 16 h. Line 1: Supernatant. Line 2: Precipitate. Line 3: BL21 (DE3). Line 4: Engineered bacteria introduced into the plasmid. Line 5 and 6: Purified sample at 16 h. Red arrows indicate bands that may be target proteins.
D. Comparison of the concentration of the same volume protein purified solution at 12 h and 16 h. Each group selected the same batch of the same volume of three bottles of bacterial culture solution, respectively purified, the same volume of purified solution to determine the protein concentration. "**" indicates p < 0.01. MW is short for Molecular weight.

Through the previous exploration of expression conditions, we found that longer low temperature culture helped to increase the content of purified proteins containing 6x His tag. Subsequently, we controlled the time of low temperature expression at 32 h for expression and subsequent verification. The culture steps are the same as before, but the culture time at 4℃ and 160 rpm is changed to 32 h, and the culture volume is expanded to 200 mL. The process of breaking up the bacteria and purifying the protein containing 6x His tag is also the same as before. Since no purified tag is present on the MS2 CP-Spytag protein, we can only prove the presence of MS2 CP by utilizing 6x His tag on SpyCatcher-EGFP after successful covalency of the SpyTag-SpyCatcher system. The expected molecular weight of SpyCather-EGFP containing 6x His tag and the complex based on SpyCatcher-SpyTag connection were 47.4 kDa and 76.5 kDa, respectively. We conducted SDS-PAGE on the protein components of the broken cells of the engineering bacteria and the purified protein components, and further conducted Western Blot analysis on the purified components, successfully proved the existence of the target components and the correct connection of the SpyTag-SpyCatcher system.

In our project design, we plan to use this VLP as a multi-functional delivery platform. Therefore, in addition to its ability to display functional proteins on the surface, it also has the function of containing RNA with a specific 19 bp stem-loop structure . In the process of verification, we preliminarily demonstrated some interaction between RNA and MS2 CP through a simple gel retardation analysis.

By reviewing the literature, we refer to a method for the preliminary detection of protein binding to nucleic acid. This method involves staining the same agarose gel with nucleic acid dyes and protein dyes. When the bands stained by both are aligned, it indicates that there is some form of interaction between the nucleic acid and the protein. It has been documented in the literature that MS2 VLP can be completely eluted during the Ni-NTA column purification process. Therefore, we mixed the purified sample which is expressed at 32 h 1:1 with TNE buffer (V/V) and then mixed it with DNA loading buffer containing glycerol for electrophoresis in 1% agarose gel. After electrophoresis, the gel was stained with 1:10,000 dilute safe red dye solution, and the results were observed and recorded. The gel was then stained with G250 dye for protein components, and the strip location was recorded after decolorization with decolorization solution. The results showed that the chromogenic site of nucleic acid was the same as that of protein and its position should be much longer than its own length, which could preliminatively indicate the existence of RNA and coat protein, so that RNA was not hydrolyzed by enzyme. Our original plan was to perform Northern Blot analysis on the samples to substantiate the findings; however, due to temporal constraints, we were unable to execute this experiment. We have therefore included it in our future validation plans.

Subsequently, we employed iodixanol density gradient centrifugation to obtain a purer concentrated solution of VLP, which was then subjected to observation via transmission electron microscopy. The samples were centrifuged at a gradient of 15%, 25%, 40%, 50%, 4℃, 36000 rpm for 4 h. The band obtained by centrifugation was absorbed, and this sample was diluted 1:1 with PBS buffer (V/V) after resuspension. Subsequently, transmission electron microscopy was performed. Correctly assembled VLP particles can be observed and the attached surface display proteins were visible on the particles.

1.The Functional Verification of Immunity Enhancers

1.1 VDAL-CPPs

In our project design, we aimed to insert the VDAL protein onto the surface of MS2-SpyTag virus-like particles (VLPs) by fusing it with SpyCatcher. To facilitate cellular entry, we added a cell-penetrating peptide (CPP) to the opposite end of VDAL, with both fusions linked by a flexible (GGGGS)4 linker. Once inserted into the VLP surface, this fusion protein would enable VLPs to enter plant cells and deliver VDAL to activate plant ETI immunity. Additionally, we designed a SpyCatcher-CPPs construct to compete for SpyTag binding sites on the VLP surface by adjusting its expression level, thereby controlling the binding of SpyCatcher-VDAL-CPPs and preventing excessive binding from interfering with VLP self-assembly. To validate the functionality of the SpyCatcher-VDAL-CPPs fusion protein and SpyCatcher-CPPs in our design, we constructed the following circuits for functional testing.

The design was verified as shown in Figure 12.

We constructed the plasmid vector shown in the figure using homologous recombination and a series of other methods.

The constructed plasmid vector is illustrated in Figure 13.

1.1.1 Transformation

After chemical transformation using calcium chloride, the constructed plasmid was introduced into E. coli BL21 (DE3). The transformed cells were plated on LB agar plates supplemented with kanamycin and incubated at 37℃ for 16 hours. Colony PCR was performed on individual colonies to verify the presence of the plasmid.

The results of the colony PCR are presented in Figure 14.

After sequencing the selected single colonies and confirming the results, the researchers proceeded with subsequent experiments.

1.1.2 SDS-PAGE

SDS-PAGE detection of bacterial total protein

1) The single colony confirmed by sequencing was inoculated into a shake flask and cultured at 37°C for 6 hours until the OD₆₀₀ reached 0.6.

2) IPTG was then added to a final concentration of 1 mM, and the culture was further incubated at 16°C for 18 hours.

3) Cells were harvested by centrifugation at 8000 rpm, 4°C for 10 minutes, washed with PBS, resuspended, and centrifuged again.

4) Finally, the cell pellet was resuspended in 1 mL of PBS to obtain a cell suspension.

5) The cell suspension was mixed with 5X Protein Loading Buffer and heated at 95°C for 10 minutes.E. coli BL21 (DE3) harboring the empty pET28a plasmid was processed in the same manner as a control.

6) Samples were analyzed by 12% SDS-PAGE, and a protein band corresponding to the expected size of SpyCatcher-VDAL-CPPs + SpyTag-6*His protein (53.7 kDa) was observed.

The results are presented in Figure 15.

Purification and SDS-PAGE analysis of the target protein

1) The single colony confirmed by sequencing was inoculated into a shake flask and cultured at 37°C for 6 hours until the OD₆₀₀ reached 0.6.

2) IPTG was then added to a final concentration of 1 mM, and the culture was further incubated at 16°C for 18 hours.

3) Cells were harvested by centrifugation at 8000 rpm, 4°C for 10 minutes, washed with PBS, resuspended, and centrifuged again.

4) Finally, the cell pellet was resuspended in 2 mL of Binding Washing Buffer (Sangon Biotech, Shanghai, China) containing 10 mM imidazole and 80 μL of PMSF (Sangon Biotech, Shanghai, China).

5) The cell suspension was then sonicated for 10 minutes with a 1-second pulse followed by a 2-second pause.

6) The cell lysate was centrifuged at 12000 rpm at 4°C for 15 minutes.

7) The supernatant was transferred to a new Eppendorf tube, and the pellet was resuspended in 1 mL of PBS for storage.

8) The supernatant was mixed with an equal volume of Binding Buffer to prepare the sample.

9) The storage solution was slowly drained, and the Ni column was equilibrated with 5 mL of Washing Buffer.

10) The sample was loaded onto the column in two bed volumes, and the first flow-through was reloaded.

11) The column was washed with two bed volumes of Washing Buffer, and the flow-through was collected until the absorbance at 280 nm reached the baseline.

12) The protein was eluted with two bed volumes of Elution Buffer, collecting 2 mL fractions each time, until the absorbance at 280 nm reached the baseline.

13) The purified protein was obtained. (Specific experimental procedures were followed from HyPur T Ni-NTA 6FF (His-Tag) PrePacked Gravity Column Kit, Sangon Biotech, Shanghai, China)

14) The harvested bacterial cell pellet, the supernatant after cell lysis, the purified protein sample, and the E. coli BL21 (DE3) cell suspension containing the empty pET28a plasmid were mixed with 5X Protein Loading Buffer and heated at 95°C for 10 minutes.

15) Samples were separated by 12% SDS-PAGE and stained with Coomassie Brilliant Blue G250 for 12 hours, followed by destaining.

The obtained results are shown in Figure 16.

The bands labeled 1, 2, 3, and 4 in the figure represent the elution fractions obtained sequentially using Elution Buffer.

The relatively large size of the target protein, which was tagged with His using the SpyCatcher-SpyTag (SpyC/SpyT) system, resulted in weaker binding affinity to the Ni-NTA column. To ensure protein purification, a Binding Wash Buffer with a reduced imidazole concentration was used. However, this led to increased contamination by non-specific proteins, although three distinct bands could still be observed near the expected molecular weight.

1.1.3 Western Blot

80 μL of each purified sample was mixed with 20 μL of 5x protein loading buffer and incubated at 98°C for 15 minutes. Samples were then loaded onto an SDS-PAGE gel and electrophoresed at 80V for 2 hours. Proteins were transferred onto a nitrocellulose membrane overnight at 4°C. The membrane was blocked and then incubated with a primary antibody (mouse anti-His-tag monoclonal antibody). After washing, the membrane was incubated with a secondary antibody (goat anti-mouse IgG). Following washing, the membrane was developed using a chemiluminescent substrate, and the results were visualized by exposure to film.

The obtained results are shown in Figure 17.

The bands labeled 1, 2, 3, and 4 in the figure represent the elution fractions obtained sequentially using Elution Buffer.
The relatively high level of contaminating proteins in the purified sample, likely due to the His-tag, suggests a potential limitation of the system. However, the presence of three distinct bands near the expected molecular weight confirms the functionality of the SpyCatcher-SpyTag (SpyC/SpyT) system. The lower intensity of the target protein band may be attributed to the relatively large size of the SpyCatcher-VDAL-CPPs fusion protein and its lower expression level.

1.2 Splip-VDAL

1.2.1 Plasmid Construction

Through the homologous recombination, a expression vector(Figure 18.) is constructed based on the shuttle plasmids pHT254, which displayed resistance to different antibiotics in E. coli DH5α and B. subtilis 168, providing a great convenience in selecting of positive colonies.

1.2.2 Protein expression analysis

To demonstrate the expression of Nlp20 and VDAL specific in Bacillus subtilis, the above plasmid vectors were transformed into B. subtilis by electrotransformation, and Splip-Nlp20 and Splip-VDAL were constitutively expressed.

To study the expression of heterologous proteins, 200mL of splip-nlp20-6*his+splip-vdal-6*his (pHT254) carried Bacillus subtilis was subjected to total-protein SDS-PAGE. The bacterial solution was centrifuged at 8000 rpm for 10 min at 4°C, and after incubating the precipitate with lysozyme at 37 °C for 1 h, the cells were sonicated for fragmentation at 300W for 10 s followed by 10 s rest. In order to achieve better lysis results, the alkaline lysis method was also used with the addition of appropriate amounts of SDS and NaOH after lysates incubating with DNase/RNase for 10 min. The lysate was centrifuged at 12000 rpm for 15min at 4°C, and the supernatant was removed to obtain the total protein sample.

Using 12% separating gel and 6% concentrating gel, the total proteins of the obtained engineered and control bacteria were subjected to SDS-PAGE, and the results are shown in Figure 19.. The VDAL and Nlp20 still carried the Splip tag because the sample was extracted from the bacterial body. Compared with the control, total protein of B.subtilis carried Splip-Nlp20-6*His+Splip-VDAL-6*His (pHT254) had a clear band at about 36 kDa, which was consistent with the size of Splip-VDAL, which weighs 36.4 kDa. However, Splip-Nlp20 was not detected in SDS-PAGE results because Splip-NLp20 was too small, which weighs 7.1 kDa.

1.3 VDAL-His

Due to the difficulty in purifying a sufficient amount of Splip-VDAL protein from the supernatant of shake flask cultures of engineered bacteria expressing Splip-VDAL, we constructed a VDAL-6*His expression system under the control of the lactose operon and T7 promoter to verify its ability to induce plant PTI responses.The design was verified as shown in Figure 20.

The constructed plasmid vector is illustrated in Figure 21.

1.3.1 Experimental Verification

Transformation

After chemical transformation using calcium chloride, the constructed plasmid was introduced into E. coli BL21 (DE3). The transformed cells were plated on LB agar plates supplemented with kanamycin and incubated at 37℃ for 16 hours. Colony PCR was performed on individual colonies to verify the presence of the plasmid.

The results of the colony PCR are presented in Figure 22.

After sequencing the selected single colonies and confirming the results, the researchers proceeded with subsequent experiments.

SDS-PAGE

Purification and SDS-PAGE analysis of the target protein

1) The single colony confirmed by sequencing was inoculated into a shake flask and cultured at 37°C for 6 hours until the OD₆₀₀ reached 0.6.

2) IPTG was then added to a final concentration of 1 mM, and the culture was further incubated at 16°C for 18 hours.

3) Cells were harvested by centrifugation at 8000 rpm, 4°C for 10 minutes, washed with PBS, resuspended, and centrifuged again.

4) Finally, the cell pellet was resuspended in 2 mL of Binding Washing Buffer (Sangon Biotech, Shanghai, China) containing 10 mM imidazole and 80 μL of PMSF (Sangon Biotech, Shanghai, China).

5) The cell suspension was then sonicated for 10 minutes with a 1-second pulse followed by a 2-second pause.

6) The cell lysate was centrifuged at 12000 rpm at 4°C for 15 minutes.

7) The supernatant was transferred to a new Eppendorf tube, and the pellet was resuspended in 1 mL of PBS for storage.

8) The supernatant was mixed with an equal volume of Binding Buffer to prepare the sample.

9) The storage solution was slowly drained, and the Ni column was equilibrated with 5 mL of Washing Buffer.

10) The sample was loaded onto the column in two bed volumes, and the first flow-through was reloaded.

11) The column was washed with two bed volumes of Washing Buffer, and the flow-through was collected until the absorbance at 280 nm reached the baseline.

12) The protein was eluted with two bed volumes of Elution Buffer, collecting 2 mL fractions each time, until the absorbance at 280 nm reached the baseline.

13) The purified protein was obtained. (Specific experimental procedures were followed from HyPur T Ni-NTA 6FF (His-Tag) PrePacked Gravity Column Kit, Sangon Biotech, Shanghai, China)

14) The harvested bacterial cell pellet, the supernatant after cell lysis, the purified protein sample, and the E. coli BL21 (DE3) cell suspension containing the empty pET28a plasmid were mixed with 5X Protein Loading Buffer and heated at 95°C for 10 minutes.

15) Samples were separated by 12% SDS-PAGE and stained with Coomassie Brilliant Blue G250 for 12 hours, followed by destaining.

The obtained results are shown in Figure 23.

The first attempt might have resulted in the target protein being included in inclusion bodies rather than appearing in the purified protein sample due to the induction conditions. To address this, the induction method was modified as follows: cells were cultured at 37°C in shake flasks for 6 hours until the OD600 reached 0.6, then IPTG was added to a final concentration of 0.1 mM, and the culture was continued at 16°C for 12 hours. Samples were prepared following the aforementioned protocol.

The obtained results are shown in Figure 24.

As shown in the figure, the target protein VDAL-6*His is clearly located in the region corresponding to its molecular weight.

1.3.2 DAB-based ROS assay

We used DAB staining kit to stain Arabidopsis leaves under different treatments.The staining results are shown in Figure 25.

The acquired images were imported into ImageJ software and converted to 8-bit grayscale images. Subsequently, the images were inverted. Given that DAB staining results in the deposition of brown-colored precipitates in regions with reactive oxygen species (ROS), with the intensity of the color directly correlating with ROS levels, the inverted images displayed lighter regions corresponding to higher ROS content.

The inverted images are presented in Figure 26.

Then we using the grayscale measurement tool in ImageJ, the contours of each leaf were outlined, and the average grayscale value for each leaf was calculated. After data normalization and analysis.

Results

The statistical results are presented in Figure 27.

Analysis of the images revealed that VDAL-CPPs or VDAL-6*His significantly induced the production of reactive oxygen species (ROS). Furthermore, the co-incubation of SA and VDAL-CPPs or VDAL-6*His resulted in a notable interference in their respective abilities to induce ROS.

2.The The Functional Verification of Cell-Penetrating Peptides(CPPs)

To validate the cell-penetrating function of the R9-Tag, commonly used in plant cells, we constructed a fusion protein of AmCyan and R9-Tag using the pET28A vector. The expression of these components is driven by the T₇ promoter under the control of the lac operon.

The design was verified as shown in Figure 28.

The constructed plasmid vector is illustrated in Figure 29.

2.1 Transformation

After chemical transformation using calcium chloride, the constructed plasmid was introduced into E. coli BL21 (DE3). The transformed cells were plated on LB agar plates supplemented with kanamycin and incubated at 37℃ for 16 hours. Colony PCR was performed on individual colonies to verify the presence of the plasmid.

The results of the colony PCR are presented in Figure 30.

After sequencing the selected single colonies and confirming the results, the researchers proceeded with subsequent experiments.

2.2 Crude extraction of AmCyan-CPPs protein

1) The single colony confirmed by sequencing was inoculated into a shake flask and cultured at 37°C for 6 hours until the OD₆₀₀ reached 0.6.

2) IPTG was then added to a final concentration of 1 mM, and the culture was further incubated at 16°C for 18 hours.

3) Cells were harvested by centrifugation at 8000 rpm, 4°C for 10 minutes, washed with PBS, resuspended, and centrifuged again.

4) Finally, the cell pellet was resuspended in 2 mL of Bacterial lysis buffer (Sangon Biotech, Shanghai, China) containing 10 mM imidazole and 80 μL of PMSF (Sangon Biotech, Shanghai, China).

5) The cell suspension was then sonicated for 10 minutes with a 1-second pulse followed by a 2-second pause.

6) The cell lysate was centrifuged at 12000 rpm at 4°C for 15 minutes.

7) The supernatant was transferred to a new Eppendorf tube.

8) Select two Arabidopsis thaliana (Columbia ecotype) plants at an appropriate growth stage. Carefully wash the roots with physiological saline solution and gently remove any debris using tweezers. Take care not to damage the roots.

9) Cut the root system at the junction of the stem and root for later use.

The supernatant and treated root system after cell lysis and centrifugation are shown in Figure 31.

10) Root tissues were immersed in protein sample and incubated at room temperature with shaking for 16 hours.

11) After being washed repeatedly more than 10 times with physiological saline under shaking conditions, no fluorescence was observed in the slide prepared with the final wash solution under a fluorescence microscope.

12) A portion of the root tissue was prepared as a water mount and observed under the Leica TCS SP8 X confocal microscope (Exλ=453nm, Emλ=484-494nm).

The obtained results are shown in Figure 32.

In the above images, we observed relatively dense fluorescent signals throughout most plant tissues, including root hair cells. To rule out the possibility that the observed fluorescence was due to the adsorption of AmCyan-CPPs proteins to the plant cell wall, the remaining root tissues after washing were used to prepare protoplasts.

The reagents used by our team to prepare protoplasts are as follows:

(1)Enzyme Digestion Buffer:

100 mM KCl
20 mM MgCl₂
20 mM CaCl₂
0.1% (w/v) Bovine Serum Albumin (BSA)
80 mM 2-(N-morpholino)ethanesulfonic acid (MES)
0.6 M Mannitol
pH adjusted to 5.5 with 0.1 M Tris-HCl

(2)Washing/Protoplast Buffer:

100 mM KCl
20 mM MgCl₂
0.1% (w/v) BSA
0.4 M Mannitol

(3)Enzyme Mixture:

1.5% Cellulase R-10 (YAKULT HONSHA CO., LTD)
0.1% Pectinase (From Aspergillus niger, Sigma-Aldrich (Shanghai) Trading Co.Ltd)

After washing, the root tissues were cut into small segments and incubated in an enzyme digestion buffer containing cellulase and pectinase at 28°C for 2 hours. The digestion process was conducted in the dark with gentle shaking to facilitate protoplast release. The digested tissue was then filtered through a nylon mesh to remove undigested tissue debris. The protoplasts were washed twice with washing buffer and centrifuged at 100 g for 2 minutes to remove cell debris. Finally, the protoplasts were resuspended in protoplast buffer. The prepared protoplasts were then observed using a Leica TCS SP8 X confocal microscope.

The obtained results are shown in Figure 33.

Based on the images, we can clearly observe a significant amount of aggregated fluorescent signals in the protoplasts, further confirming the efficient membrane penetration of R9 cell-penetrating peptide into plant cells.

Cry6Aa2 toxin protein validation results

To verify the expression of Cry6Aa2 protein, we fused a 6×His label to the C-terminal of the protein for purification, and controlled the expression by lactose operon lacI. cry6Aa2 gene with 6*His tag was cloned into pET28a plasmid and transformed into E. coli BL21(DE3) for expression.

The results of agarose gel electrophoresis of colony PCR products demonstrated that Cry6Aa2 expression vector was successfully constructed.

E. coli BL21 (DE3), which transformed pET28a-Cry6Aa2 expression vector, was cultured at 37°C to OD600 of 1.0, and then induced to express with 1 M IPTG for 8 hours. After the bacteria were collected, the bacteria were ultrasonically lysed and the total proteins were purified. SDS-PAGE and Western Blot analysis confirmed the successful expression of Cry6Aa2 protein.

We then built a vector that included the SpyCatcher/SpyTag system. Cry6Aa2 with CPPs and SpyCatcher were linked into a fusion protein through (GGGGS) ₄, and the gene of the fusion protein was recombined into pET28a vector containing SpyTag.

E. coli BL21 (DE3) with recombinant plasmid was cultured. After 12 h of culture, the bacteria were enriched and lyzed, and the total protein of the bacteria was purified. The total protein of the bacteria and the purified sample were conducted on SDS-PAGE.

The weight of the complex protein is 68.3 kDa, which can be observed on the SDS-PAGE.So,The complex protein was successfully expressed.

Validation of TAA Secretion Function

A reombinant vector carrying tbrA and tbrB was transfered into B.subtilis. The engineered B. subtilis can express TbrA/B protein to produce and secrete trans-aconitoic acid(TAA).In order to verify the ability of engineered bacteria to secrete TAA, we fermented 200 mL of engineered bacteria culture medium for 24 h and 32 h, respectively, and crude TAA was extracted from the supernatant and tested by HPLC. The results are as followed.

16D10 shRNA design

We designed shRNA targeting the 16D10 gene encoding parasitic peptides of nematodes to reduce the infestation of plants by nematodes.

Bacillus mainly relies on the assembly of TasA into amyloid fibrillary structures to form biofilms, while also promoting biofilm formation by producing major and minor wall teichoic acid (WTA).TapA is a protein of the genus Bacillus that promotes the formation of its biofilm by promoting the assembly of TasA into amyloid fibrillary structures.GgaA is a minor wall teichoic acid (WTA) synthase contained in the genus Bacillus that promotes the formation of Bacillus biofilm by synthesizing minor WTA.

We selected the pHT254 shuttle plasmid as the vector, which is a plasmid that can replicate in both E. coli and Bacillus subtilis. Then, We constructed two different circuits, one can only increase the expression level of TapA and the other one can increase the expression level of TapA and GgaA at the same time.

Firstly, we successively transformed E. coliDH5α with two plasmids. After colony PCR results and gene sequencing were verified by agarose gel electrophoresis, we believed that two different plasmids were successfully obtained. The plasmid was then extracted from the successfully transformed E. coil DH5α and used for the transformation and functional verification of B. subtilis 168.

Then we transferred the extracted plasmids into B. subtilis168 through electrical transformation, and through the results of generation sequencing, we successfully obtained two different engineered bacteria, GT(l) and GT(h).

Firstly, the proteins expressed by GT(l) and GT(h) were purified and separated respectively, followed by SDS-PAGE analysis with the whole proteins extracted from the unconverted B. subtilis168 (Figure 45). As can be seen from the figure, GT(l) only expresses TapA, while GT(h) expresses both GgaA and TapA.

Then, by incubating GT(l) and untransformed B. subtilis168 in separate wells of a 96-well plate at a constant temperature to produce biofilms, we fixed the biofilms and stained them with 1% crystal violet solution, then dissolved the crystal violet with 33% acetic acid solution and measured the A₅₉₀ values of the different groups dissolved crystal violet (Figure 46).

Three parallel repeated experiments were conducted in each group at the same time, and the results showed that increasing the expression level of TapA could significantly improve the biofilm generation ability of B. subtilis168.

Finally, We incubated GT(h/l) and untransformed B. subtilis 168 in separate wells of a 96-well plate at a constant temperature to produce biofilms, and used the same method to quantitatively detect the biofilm(Figure 48).

Six parallel repeated experiments were conducted in each group at the same time, and the results showed that increasing the expression level of TapA could significantly improve the biofilm generation ability of B. subtilis 168, and this effect was more significant after increasing the expression level of GgaA and TapA at the same time.

In order to release VLP smoothly into the extracellular, we designed a lytic release module.To ensure that the toxic-related proteins do not express until after VLP synthesis and assembly is complete, we designed a switch based on an AND door. Figure 50. Designed lysis circuit.

The final proteins in the circuit that synthesis VLP are HrpR and HrpS,once they expressed,they will form a heteromeric complex,activate the promoter P_hrpL.Lysis genes are then expressed to lyse the bacteria.

We choose two kind of proteins.One is holin (a perforin) ,another is a phage-encoded peptidoglycan hydrolases.They will trigger the cleavage of the bacteria,help to release the VLP.

To verify the effectiveness of our release system,we designed a verification circuit according to the schematic below.we choose the Xylose-inducible promoter to induce protein expression (Figure 51). Figure 51. Varified lysis circuit.

Lytic gene was combined with plasmid pHT315,which includes a set of xylose operon,so as to regulate the expression of lytic genes by D-xylose.

We first performed colony PCR on the B. subtilis 168 colony to determine whether the recombinant plasmid had entered the bacterial interior. We picked out six colonies.The results showed that we had a strain that had completed the transformation, so we began a series of induction experiments.

At first, we carried out the experiment following the operation of studying the lysis of E.coli. The B. Subtilis 168 bacterial solution containing the recombinant plasmid was divided into two parts and transferred to the bacterial bottle.One part was added with 60 mM/L D-xylose and the other was not treated.

The bacterial solution of the same recombinant strain was divided into two parts, one part was added with D-xylose (Treatment), and the other part was not treated (Control),All bacteria were cultured in 10mL bacterial vials and the OD₆₀₀ value was measured every 40 minutes for 8 consecutive times.

The initial cracking effect was not ideal, in order to make a more perfect effect, combined with the aerobic properties of B. Subtilis, we designed a new experiment.

In this experiment, all B. Subtilis 168 were placed in a bacterial vial sealed with a non-breathable membrane, and common B. Subtilis 168 was added as a control.

We've prepared two parts of solution,one part were normal B. Subtilis 168 (Control), another part were transformed B. Subtilis 168 (Treatment). Then they were added with different concentrations of xylose as shown in the figure, sealed with a breathable film, and cultured for 12 hours, after which the OD₆₀₀ value was measured.

Based on the results, it was concluded that the holin and PGHs can causing the cleavage of bacteria.This ensures that the VLP particles can be released smoothly.

Both Bacillus subtilis and Bacillus Velez are endophytic bacteria of plants, which means that they can colonize crops. Therefore, in order to avoid the leakage of engineered bacteria, we chose the light-sensitive pathway as the suicide module, that is, to kill bacterial strains entering the external environment by expressing the phototoxic protein KillerRed. This method is simple and effective, and does not impose additional financial burden on farmers.

We selected the pHT315 shuttle plasmid as the vector, which is a plasmid that can replicate in both Escherichia coli and Bacillus subtilis. At the same time, due to the phototoxicity of KillerRed, we chose the xylose operator as the promoter, which allows us to induce expression at the right time in order to perform accurate phenotypic experiments and reduce the interference of external factors. Finally, we assembled the part by In-Fusion cloning and imported Bacillus subtilis B. subtilis168 by electrical transformation. The part has been verified by sequencing.

We then conducted a preliminary induction and test on this part: we set up an induction group and a blank group, added 60 mM xylose in the induction group, and added an equal amount of sterile water in the blank group. After 20h of induction expression, the color of the bacterial solution in the two groups was observed and compared with the result of irradiation under sunlight (light intensity = 5000 lux) for 2h.

The experimental results effectively proved that our recombinant vector successfully expressed KillerRed and played a full suicidal role in Bacillus subtilis. But at the same time, we noticed that there was a big gap between the Control group and the Treated group in the initial OD₆₀₀, and the Control group also died under natural light. The former may be due to the growth burden of premature induction expression on the Treated group. The latter may be due to the ultraviolet light in the sun itself has a certain bactericidal effect.

Therefore, we made further improvements to the experimental conditions and conducted more in-depth tests. We first induced the KillerRed in the same way, performing a 3-hour light experiment at OD₆₀₀=1 to test the sensitivity of Killerred to different colors of light and compare OD₆₀₀ growth over that time. Figure 60. The chang of bacterial culture OD₆₀₀ change in different colors of light.
The color of the histogram corresponds to the color of the light received by the engineered bacteria, and the color corresponds to the size of the wavelength. Obviously, the engineered bacteria were most sensitive to green light, and the effect decreased with the increase and decrease of wavelength.

Then, We improved the induction conditions: cultured engineered bacteria for 4-6h until the bacterial solution was slightly cloudy, and then added 60 mM/L xylose to induce expression for 10-12h. In order to verify that KillerRed is mainly stimulated by Green Light and plays a suicidal role, we set up the Control group (Light avoidance), the green light group (artificial green light irradiation, light intensity =3000 lux) and the Natural Light group (natural light irradiation, light intensity = 3000 lux). Light intensity =30000 lux) compared with each other, the experimental results are as follows:

Control group: Treated with Light avoidance.Green Light group:Treated with artificial green light.Natural Light group:Treated with natural light for a total of 3 hours.

As shown in the figure, KillerRed has a very obvious killing activity in Bacillus subtilis, and this effect is mainly brought about by green light excitation.