The salicylic acid biosensor is composed of the NahR and the corresponding sal promoter[1]. When NahR binds SA, the P_sal is activated.

We constructed a vector that AmCyan follows the P_sal and induced the expression with different concentrations of SA. The results are shown below.

In order to verify the self-assembly of VLP and the correct connection function of Spytag-Spycatcher system, we constructed the sequence of MS2 capsid protein containing SpyTag peptide and Spycatter-EGFP fusion protein on PUC57 mini plasmid. We purified the extracted bacterial protein and verified the expression of the protein and the correct assembly of the VLP.

We first performed SDS-PAGE verification on the extracted and purified bacterial proteins from BL21 (DE3) strain that transformed the plasmid. 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 Spycatch-EGFP after successful covalency of the SpyTag-SpyCatcher system[2]. 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.

In order to further confirm that the band is the target protein, Western Blot analysis was performed using antibodies against 6x His label. The location of the band was consistent with the size of the target protein, which proved the successful expression of the target protein and the correct connection of SpyTag-SpyCatcher.

After confirming the presence of correctly connected SpyCatcher and MS2 capsid protein containing SpyTag sequence in the bacteria, we isolated VLP by centrifugation of the pre-treated 1 L of expanded cultured bacterial crushing solution with a density gradient of 15%, 25%, 40% and 50% of ioxanol.

Subsequently, the isolated protein solution was diluted 1:1 with PBS buffer (V/V) and negative staining was performed. The stained samples were observed under transmission electron microscope. The TEM photographs demonstrate the successful assembly of the VLP and show the successful connection of the proteins on the surface of the VLP.
Figure 3. Density gradient centrifugation results and correct assembly of VLP particles.
A. Density gradient centrifugation results. The red arrows indicate the separated bands.
B, C. Image of correctly assembled VLP under a transmission electron microscope.
The black arrows represent proteins attached to the particle surface.

The section of plant immunity is aimed at enhancing the crops’ resistance to nematodes through triggering PTI and ETI [3][4].For use, Basilis subtilis secretory signal peptide, Splip[3], is linked to the immunity effectors.(BBa_K5335019) Additionally, cell-penetrating peptides (CPPs), SR9, are used to bring the effector into the plant cells on the VLPs.(BBa_K5335025)

The immunity enhancement has been verified through DAB staining. The result(Figure 4,6.) demonstrates that the VDAL can be expressed in bacteria and promote plant immunity.

The CPPs are proved to be applicable as well by the observation of laser confocal microscope(LCM).

First, we wanted to verify the successful expression of Cry6Aa2[5], which was first verified on Escherichia coli BL21(DE3). Cry6Aa2 tagged with 6*His was constructed onto pET28a plasmid with lactose operon.Cry6Aa2 protein was induced by IPTG, and the samples purified by Ni column were analyzed by SDS-PAGE and Western Blot.(BBa_K5335006)

SDS-PAGE and Western Blot results (Figure nn) demonstrated that Cry6Aa2 was successfully expressed in Escherichia coli BL21(DE3). In SDS-PAGE results, there were thicker bands in the induced group than in the non-induced group at 49 kDa. However, the uninduced group also showed bands at 49 kDa, so we further demonstrated the expression of Cry6Aa2 by Western Blot. Western Blot results showed that, compared with the non-induced group, there were thicker bands near 49 kDa, and the depth of the bands was significantly different, but there were still shallower bands at 49 kDa in the non-induced group, which was speculated to be caused by low dose leakage of lactose operon. In conclusion, we successfully expressed Cry6Aa2 protein.

Secondly, a reombinant vector carrying tbrAandtbrB was transfered into B.subtilis[6]. The engineered Bacillus 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 Figure .:

We designed a circuit to enhance the expression of GgaA and TapA proteins in Bacillus to improve biofilm formation and colonization capabilities[7][8]. By using a strong promoter and RBS, two different circuits were constructed and successfully transformed into the B.subtilis 168 strain to verify the system's efficacy.

By incubating two different engineered bacteria and untransformed B.subtilis 168 in different wells of the same 96-well plate at a constant temperature to form biofilms, the biofilms were fixed and stained with 1% crystal violet. After dissolving the crystal violet with a 33% glacial acetic acid solution, the A₅₉₀ of the dissolved crystal violet was measured for different groups.

The results indicate that B.subtilis 168 with both genes introduced formed the thickest biofilm, B.subtilis 168 with only TapA introduced had the second thickest biofilm, and the WT group, which did not introduce any genes, formed the thinnest biofilm.

In order to release VLPs into the extracellular space smoothly, we choose two kind of proteins.One is holin (a perforin) ,another is a phage-encoded peptidoglycan hydrolases[9].They will trigger the lysis of the bacteria,help to release the VLP.

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.

To reflect the function of lysis circuit directly, we compared the normal B.subtilis168 with the transformed B.subtilis168, induced with different concentrations of D-xylose, and treated it in hypoxic culture bottles covered by a sealed film.

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

We combined KillerRed with pHT315 plasmid with xylose operon to form an expression whole[10], induced protein expression of xylose in Bacillus subtilis, and finally carried out our verification experiment under different light conditions.

We first tested KillerRed's sensitivity under different colors of light to find the appropriate color of excitation light and verify that KillerRed was functioning properly. We then tested KillerRed under both artificial green and natural light to prove that our device works as intended. Finally, we successfully completed the functional verification of this part, and the results are as follows:

Figure 12. Bacterial 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.
Figure 13. Bacterial OD₆₀₀ change over illumination time. The Control group was treated with Light avoidance, the Green Light group was treated with artificial green light, and the Natural Light group was treated with natural light for a total of 3 hours.