iGEM 2024 — Team hzau-china

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

{% endblock %}

Engineering

![](https://static.igem.wiki/teams/5335/description/tree.png)

Circuit Iteration
Salicylic Acid Biosensor
VLP Delivery System
Plants Immunity Enhancement
Nematode Killing Module
Colonization
Lysis & Release Module
Suicide
Reference

Initial Circuit Design

In our initial circuit, we employed *B. subtilis* as the chassis organism. Upon salicylic acid induction[1], the downstream circuit was activated, leading to the expression of a SpyCatcher-Cry6Aa2 fusion protein and an shRNA containing an MS2 stem-loop[2][3][4]. The MS2 RNA stem-loop facilitated self-assembly of the MS2 capsid protein[5]. Subsequently, SpyTag covalently linked with SpyCatcher-Cry6Aa2, forming complete virus-like particles (VLPs). Through a cascade reaction, the two-component system HrpR and HrpS induced the expression of a lysis enzyme, resulting in the release of VLPs and subsequent killing of nematodes[6].

![](https://static.igem.wiki/teams/5335/design-box11/1.png)
 **Figure 1. Circuit improvement on March 15, 2024.**

## Cycle 1

Given the robust nature of nematodes, our instructor suggested incorporating a plant immunity module. By enhancing plant disease resistance, we aim to reduce nematode damage and invasion.

Improvements: Induced by L-Ala and regulated by FapR, the bacteria can produce plant immunity-activating proteins VDAL and NLP20 fused with the exosomal tag Splip[7][8][9]. These proteins can also be induced by salicylic acid through a cascade reaction.

![](https://static.igem.wiki/teams/5335/design-box11/2.png)
 **Figure 2. Circuit improvement on March 23, 2024.**

## Cycle 2

We incorporated SpyCatcher-VDAL-CPPs(R9) into the circuit to enable its entry into plant cells and activation of the plant ETI response[10]. Concurrently, to enhance the delivery of Cry6Aa2 protein into nematodes, we also added a CPPs tag to the circuit[11].

![](https://static.igem.wiki/teams/5335/design-box11/3.png)
 **Figure 3. Circuit improvement on March 28, 2024.**

## Cycle 3

To enhance nematode killing efficacy, we incorporated a TAA production and transport module based on our literature review demonstrating the nematicidal activity of TAA molecules[12].

![](https://static.igem.wiki/teams/5335/design-box11/4.png)
 **Figure 4. Circuit improvement on March 31, 2024.**

## Cycle 4

To enhance the colonization ability of our engineered strain, we found in the literature that the TapA gene can promote biofilm formation and adhesion by increasing amyloid protein expression in the *B. subtilis* cell wall, thereby providing better root protection[13].

![](https://static.igem.wiki/teams/5335/design-box11/5.png)
 **Figure 5. Circuit improvement on April 2, 2024.**

## Cycle 5

To enhance the biosafety of our engineered strain, we incorporated a suicide module and a light-inducible toxic protein, KillerRed, to mitigate the risk of environmental release[14].

![](https://static.igem.wiki/teams/5335/design-box11/6.png)
 **Figure 6. Circuit improvement on April 5, 2024.**

## Cycle 6

Following our advisor's suggestion, we have transitioned from using a single Bacillus velezensis strain to a consortium of two species, including Bacillus subtilis. The functions of the two strains have been redistributed: *B. subtilis* primarily colonizes the leaf surface and activates plant immunity, while B. velezensis primarily colonizes the root and is responsible for rhizosphere protection and nematode killing. We have named this project "Bacillus Binary Star." In this optimization, considering the uncertainty of the L-Ala induction module, we have removed it and replaced all inductions with salicylic acid-dependent systems.

![](https://static.igem.wiki/teams/5335/design-box11/7.png)
 **Figure 7. Circuit improvement for Bacillus velezensis on May 15, 2024.**

![](https://static.igem.wiki/teams/5335/design-box11/8.png)
 **Figure 8. Circuit improvement for Bacillus subtilis on May 15, 2024.**

## Cycle 7

A holin (Holin) and an endolysin (PGHs) derived from bacteriophage VB\_sup-GOe1 have been introduced to replace the enterococci-derived lysin, providing a more targeted and efficient lysis system for Bacillus species[15] .

![](https://static.igem.wiki/teams/5335/design-box11/9.png)
 **Figure 9. Circuit improvement on May 12, 2024.**

## Cycle 8

We have introduced SA-inducible SpyCatcher-CPPs into both engineered strains to regulate the binding of SpyCatcher-VDAL-CPPs to the VLP surface. By adjusting the expression levels of SpyCatcher-CPPs, we can fine-tune the binding affinity and prevent excessive binding that may compromise the VLP's self-assembly and function.

![](https://static.igem.wiki/teams/5335/design-box11/10.png)
 **Figure 10. Circuit improvement on May 15, 2024.**

## Cycle 9

Although TapA enhanced biofilm formation, the results were not as robust as expected. Therefore, we introduced, a wall teichoic acid synthase[16], into the circuit to further enhance biofilm expression. Given that the flagellum gene, flg, primarily regulates nematode motility, we streamlined the circuit by reducing its expression to ensure more effective nematode inhibition.

![](https://static.igem.wiki/teams/5335/design-box11/11.png)
 **Figure 11. Circuit improvement on July 12, 2024.**

## Final Design

Through continuous iteration, we have refined our designs, significantly reducing the waste of experimental resources and failures caused by circuit design flaws.

By iteratively refining our designs, we have effectively minimized the experimental failures and resource wastage stemming from circuit design issues.

![](https://static.igem.wiki/teams/5335/design-box11/12.png)
 **Figure 12. Final design for Bacillus velezensis.**

![](https://static.igem.wiki/teams/5335/design-box11/13.png)
 **Figure 13. Final design for Bacillus subtilis.**

Salicylic Acid Biosensor

## Cycle 1

### Design

The first step in our engineered genetic circuit is to activate the LysR-type transcriptional regulator NahR with the sal promoter. We aim to construct a salicylic acid biosensor on a plasmid and use synthetic SpyCatcher-EGFP-6\*His as a reporter gene for functional validation.

### Build

Due to the assembly of NahR and the sal promoter onto two separate plasmids during gene synthesis, we aim to recombine them into a single pUC57-mini plasmid. By utilizing the SpyCatcher-EGFP-6\*His reporter gene downstream of the sal promoter, we intend to transform the construct into E. coli BL21(DE3) for functional validation. The entire process involves two rounds of In-Fusion cloning. As the first step, we will perform In-Fusion cloning to integrate NahR into the pUC57 plasmid.

![](https://static.igem.wiki/teams/5335/nahr1/1.png)
 **Figure 14. Flowchart for constructing NahR into the pUC57 plasmid.**

Agarose gel electrophoresis of colony PCR products showed that we successfully constructed the expression vector.

![](https://static.igem.wiki/teams/5335/nahr1/2.png)
 **Figure 15. Direct translation: Results of 1.5% agarose gel electrophoresis for colony PCR.**

M: DL2000 DNA Marker; 1-5:Five single colonies obtained after In-Fusion cloning.

We confirmed the successful integration of NahR into the pUC57 plasmid by colony PCR, followed by transformation into E. coli DH5α. Subsequently, we employed In-Fusion cloning to insert the fragment containing the sal promoter and EGFP-6\*His into the aforementioned plasmid.

![](https://static.igem.wiki/teams/5335/nahr1/3.png)
 **Figure 16. Flowchart for constructing EGFP-6\*His into the pUC57 plasmid.**

Agarose gel electrophoresis of colony PCR products showed that we successfully constructed the expression vector.

![](https://static.igem.wiki/teams/5335/nahr1/4.png)
 **Figure 17. Direct translation: Results of 1.5% agarose gel electrophoresis for colony PCR.**

M: DL2000 DNA Marker; 1-12:Twelve single colonies obtained after In-Fusion cloning.

Colony PCR confirmed the successful integration of the fragment containing the sal promoter and EGFP-6\*His into the previously constructed plasmid, which was then transformed into E. coli DH5α.

![](https://static.igem.wiki/teams/5335/nahr1/5.png)
 **Figure 18. Sequencing results confirming the construction of the plasmid in Cycle 1.**

Sanger sequencing verified the accuracy of the constructed plasmid, ensuring the absence of mutations that could compromise experimental results.

### Test

The constructed plasmid was extracted and transformed into E. coli BL21(DE3) for validation. A preliminary experiment was conducted by inducing with salicylic acid to a final concentration of 500 μM. After 6 hours of induction, the fluorescence intensity was measured using a microplate reader, but no significant difference was observed compared to the uninduced group.

### Learn

Analysis suggests that the fusion with EGFP may have compromised its fluorescence properties. Therefore, we plan to use amCyan, a fluorescent protein successfully validated by our team in 2023, for further verification.

## Cycle 2

### Design

The characterized cyan fluorescent protein from 2023, which was assembled into the pFF745 plasmid, was used to replace EGFP-6\*His in the pUC57 plasmid via In-Fusion cloning

### Build

We replaced EGFP-6\*His, which was constructed in Cycle 1, with the cyan fluorescent protein amCyan in the pUC57 plasmid using In-Fusion cloning.

![](https://static.igem.wiki/teams/5335/nahr1/6-1.png)
 **Figure 19. Flowchart for replacing EGFP-6\*His with amCyan on the pUC57 plasmid.**

![](https://static.igem.wiki/teams/5335/nahr1/6.png)
 **Figure 20. Results of plasmid construction in Cycle 2.**

A.Results of 1.5% agarose gel electrophoresis of colony PCR products from Cycle 2 plasmid construction. M: DL1000 DNA Marker. 1-8: Five individual colonies obtained after In-Fusion cloning.B.Sequencing results of the plasmid constructed in Cycle 1.

Colony PCR confirmed that amCyan had successfully replaced EGFP-6\*His in the plasmid, and the resulting construct was successfully transformed into E. coli DH5α. Sequencing results verified the correctness of the construct.

### Test

When we transformed the plasmid into E. coli BL21(DE3) cells, we observed significant fluorescence in the uninduced culture, even without the addition of salicylic acid.

![](https://static.igem.wiki/teams/5335/nahr1/7.png)
 **Figure 21. Results of the phenotypic preliminary experiment of the gene circuit constructed in Cycle 3.**
The weight of the target protein is 68.3 kDa.Ctrl:Empty *E.coli* BL21(DE3). Ex. *E.coli* BL21(DE3) that has been introduced into the expression vector.1 and 2 were purified samples.

### Learn

We have proposed the following hypothesis to explain this phenomenon: the endogenous salicylic acid in E. coli is sufficient to activate the sal promoter, thereby inducing the expression of downstream amCyan. However, this conclusion contradicts the results we found in the literature. Upon further investigation of the literature and iGEM projects, we discovered that the salicylic acid sensor in the part BBa\_J61051 was not characterized in more detail. Due to the absence of approximately 40 bp of upstream regulatory sequence in the Psal sequence we used, the repressor NahR cannot exert its inhibitory effect.

## Cycle 3

### Design

Based on the problems found in Cycle 2, we decided to add the NahR binding sequence upstream of the sal promoter using overlap extension PCR.

### Build

Using the plasmid constructed in Cycle 3 as a template, we added a 40 bp NahR binding sequence via overlap extension PCR. After PCR, the template plasmid was digested with DpnI, a restriction endonuclease that specifically recognizes the 5'-Gm6ATC-3' site. The digested plasmid was then transformed into E. coli DH5α. Due to the short 40 bp insertion, colony PCR was not feasible for differentiation. Therefore, we directly picked individual colonies and sequenced them.

![](https://static.igem.wiki/teams/5335/nahr1/8.png)
 **Figure 22. Cycle 3 molecular cloning results.**

A. Plasmid map with added NahR binding sequence. B. Sequencing results of the plasmid with added NahR binding sequence.

Sequencing results confirmed the successful insertion of a 40 bp NahR binding sequence upstream of the sal promoter.

### Test

Subsequently, we attempted induction with 500 μM salicylic acid. Interestingly, we observed a striking green fluorescence in the uninduced group from the same colony, while the groups induced with 500 μM salicylic acid and sodium salicylate exhibited no fluorescence.

![](https://static.igem.wiki/teams/5335/nahr1/9.png)
 **Figure 23. Phenotype of the gene circuit constructed in Cycle 3.**

### Learn

We speculate that the leaky expression in the uninduced group might be due to the proximity of the previous transcription unit to the NahR binding sequence. The stem-loop structure in the rrnB T1 terminator might have inhibited the formation of the stem-loop induced by NahR.

## Cycle 4

### Design

Considering the issues identified in Cycle 3, we reversed the orientation of the NahR transcription unit and conducted subsequent validation experiments.

### Build

We inverted the orientation of the NahR transcription unit using In-Fusion Cloning to construct the plasmid shown in the figure.

![](https://static.igem.wiki/teams/5335/nahr1/10.png)
 **Figure 24. A flowchart for the reverse of the NahR transcriptional unit on the pUC57 plasmid.**

![](https://static.igem.wiki/teams/5335/nahr1/11.png)
 **Figure 25. Direct translation: Results of 1.5% agarose gel electrophoresis for cycle 4.**

M: DL2000 DNA Marker; 1-5:Five single colonies obtained after In-Fusion cloning.

Colony PCR results confirmed the successful construction of the plasmid with the inverted NahR transcription unit. Sequencing analysis further revealed no mutations that would affect the experimental outcome.

### Test

Subsequently, a preliminary induction experiment was conducted using 500 μM salicylic acid. The culture was incubated at 37℃, 200 rpm for 12 hours.

![](https://static.igem.wiki/teams/5335/nahr1/12.png)
 **Figure 26. Cycle 3 molecular cloning results.**

In our preliminary experiments, we verified the functionality of the salicylic acid biosensor. Subsequently, we set up induction experiments with 0 μM, 0.1 μM, 1 μM, 10 μM, 100 μM, and 1000 μM salicylic acid, as well as a negative control lacking the sal promoter. 200 μL of each culture was added to a 96-well plate, and the fluorescence intensity at 486 nm was measured using a microplate reader. Each induction concentration was tested in five biological replicates.

![](https://static.igem.wiki/teams/5335/nahr1/13.png)
 **Figure 27. Verification results graph of salicylic acid biosensor.**

### Learn

We observed a rapid increase in fluorescent protein expression when the salicylic acid concentration was between 1 and 10 μM. Moreover, compared to the negative control, the basal expression from the sal promoter was relatively low.

VLP delivery system

## Cycle - Construction of vector plasmids

### Design

In order to display our functional protein on the surface of virus-like particles (VLPs), we have selected the SpyTag-SpyCatcher system as the scaffold for protein display on VLPs after a thorough review of the literature.

We plan to fuse the coat protein of the bacteriophage MS2 of *E.coli* with SpyTag, thereby creating a SpyTag site on the surface of the coat protein capable of binding to SpyCatcher. By consulting reference literature, we identified a previously studied sequence of the MS2 coat protein fused with SpyTag and constructed it into the pUC57mini plasmid.

Initially, we plan to individually integrate spycatcher-egfp and ms2 loading sequence into a plasmid containing nahR and ms2-spytag, followed by transformation into *E.coli* to verify protein expression, VLP assembly, and validation of the encapsidation of the MS2 binding sequence. The genes mentioned above are expressed constitutively in bacteria.

### Build

We propose to utilize two rounds of homologous recombination to integrate the spycatcher-egfp with the ms2 loading sequence into the pUC57 mini plasmid vector containing the MS2 coat protein (CP) sequence. To facilitate this integration, we have engineered homologous arms flanking the spycatcher-egfp sequence and linearized the original plasmid vector. The integration was accomplished via homologous recombination, and the successful incorporation was confirmed through colony polymerase chain reaction (PCR) and subsequent plasmid sequencing (**Figure 28** to **30**).

![](https://static.igem.wiki/teams/5335/ms2-spy-e/first-infusion.png)
 **Figure 28. First homologous recombination.**

Two recombinations are planned to complete the construction.

![](https://static.igem.wiki/teams/5335/ms2-spy-e/first-bacp.png)
 **Figure 29. Colony PCR for fragments containing spycatcher-egfp.**

![](https://static.igem.wiki/teams/5335/ms2-spy-e/ms2-rna-p.png)
 **Figure 30. Colony PCR for fragments containing ms2 loading sequence.**

### Test

We submitted the assembled plasmid for sequencing and planned to validate protein expression upon successful sequencing. However, the returned results indicated a premature termination of the MS2 CP sequence following SpyTag, and the recombination of the ms2 loading sequence was unsuccessful (**Figure 31.**), necessitating the reconstruction of the vector for further experimentation. This suggests that the previous construction attempt was unsuccessful.

![](https://static.igem.wiki/teams/5335/ms2-spy-e/wrong-sequence.png)
 **Figure 31. The returned sequencing results showed significant problems.**

### Learn

The recombination process in this instance failed to construct the anticipated plasmid, likely due to: 1. The original Taq polymerase used exhibited poor fidelity, resulting in nonsense mutations. For subsequent amplification, a high-fidelity Phanta polymerase will be employed in the experiment. 2. The efficiency of homologous recombination for small fragments is low; therefore, a new strategy will be adopted to recombine larger fragments into the plasmid to complete the construction of the expression vector.

### Redesign

Due to the challenges encountered with the integration of small fragments, we have revised the sequences of the fragment and the vector, and employed a high-fidelity DNA polymerase to ensure the successful construction of the vector in this instance.

### Rebulid

Due to the challenges associated with the incorporation of small fragments, we integrated the spycatcher-egfp with the ms2 loading sequence into the same plasmid during gene synthesis (**Figure 32.**) . Concurrently, to facilitate future validation of the salicylic acid regulatory element's effect on the binding of SpyCatcher to the SpyTag on the surface of VLPs, we designed the fragment to be integrated as the nahR-ms2 cp+spytag sequence for this round. Additionally, we have reserved a site upstream of spycatcher-egfp for the insertion of the Psal promoter, which will be utilized for future validation of salicylic acid regulation. The genes mentioned above are still expressed constitutively in bacteria. Colony PCR revealed the presence of access fragments (**Figure 33.**) .

![](https://static.igem.wiki/teams/5335/ms2-spy-e/secondary-fusion.png)
 **Figure 32. Reconstructed plasmid vector.**

![](https://static.igem.wiki/teams/5335/ms2-spy-e/bl21-correct-ms.png)
 **Figure 33. Colony PCR revealed the presence of access fragments.**

### Test

Subsequently, we submitted the newly recombined vector for sequencing, and the returned results confirmed that all functional fragments are correctly present on the plasmid, indicating the successful completion of the expression vector construction (**Figure 34.**).

![](https://static.igem.wiki/teams/5335/ms2-spy-e/total-sequence.png)
 **Figure 34. Sequencing confirmed the successful assembly.**

Following the confirmation of successful plasmid construction, we transformed it into the BL21 (DE3) strain, which is utilized for protein expression. Post-extraction and purification, the correct assembly of VLPs and the binding of SpyCatcher on the surface of VLPs were observed under transmission electron microscopy (**Figure 35.**) . This observation substantiates the success of our construct.

![](https://static.igem.wiki/teams/5335/ms2-spy-e/f6.png)
 **Figure 35. Density gradient centrifugation results and correct assembly of VLP.**
**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.

### Learn

During this round of construction, we observed that the success rate of homologous recombination for larger fragments is significantly higher than that for smaller fragments. In the future, when constructing vectors using homologous recombination methods, we must also take into account the size of the recombination fragments. Should there be a need to insert specific small fragments, the success rate of constructing vectors using enzymatic restriction and ligation methods may be higher.

Plants Immunity Enhancement

### The Cycle 1

**Design**

Considering that the VLP releasing is lightly behind the initial time of plant infestation by nematodes, in order to better enhance the effect of our engineered bacteria on controlling nematodes, the project transferred two kinds of plant immune activators carrying exogenic labels of Bacillus proteins.

![](https://static.igem.wiki/teams/5335/aspf2-splip/eng-c1-db.png)
 **Figure 36. The plasmids constrution.**

A. The plasmid constructed to express the Splip-Nlp20 and Splip-VDAL in *B.subtilis*, each of the Splip-Nlp20 and Splip-VDAL is linked with a 6\*HIs-tag which is used for the future nickel ion affinity chromatography purifying. B. The history of contrsucting the plasmid, the fragment is artificial synthesis and inserted into the pHT254 backbones by homologous recombinant.

**Build**

The pHT254 was linearized by PCR and insert the fragment. The plasmids are expected to express Splip-Nlp20-His and Splip-VDAL-His, which is able to be secreted outside *B.subtilis* body.

The pHT254 was linearized by PCR. However, when the ordinary protocol was applicated and check by agarose gel electrophoresis, the results were either dispersion band or no band. In that case, a “Two-steps” method is used to gain the usable linearized pHT254 backbones. In order to reduce the occurrence of false positives, DpnI Quick Cut is used to treat the recovered linearized plasmid backbone. The fragment was inserted successfully and the initial vector was transferred in to *B.subtilis* 168.

![](https://static.igem.wiki/teams/5335/aspf2-splip/eng-c1-line.png)

**Figure 37. The linerization results of pHT254 detected by agarose gel electrophoresis.**

A, B. The dispersion band and no band result of ordianary method. c. The correct band result of the ‘two-steps’ method. M: DL10,000 DNA Marker

**Test**

After successfully construct the vector(**Figure 38. A.**), the protein function verifycation expreiments was in process.In the process of verifying the normal expression and secretion of Splip-Nlp20-His and Splip-VDAL-His, we treated 20mL of Bacillus subtilis with pHT254 in 24 h culture solution supernatant with nickel ion affinity chromatography,analyzing the purified products and unpurified culture by SDS-PAGE. It was found that the experimental groups(the unpurified culture and purified product) had a significant band at 17 kDa, which was different from the expected band of about 34 kDa(**Figure 38. B.**).

![](https://static.igem.wiki/teams/5335/aspf2-splip/eng-c1-sq.png)

**Figure 38. The DNA sequencing and SDS-PAGE of puirified product.**

A. The sequencing verification of plasmid construction, each of the Splip-Nlp20 and Splip-VDAL is linked with a 6\*HIs-tag which is used for the future nickel ion affinity chromatography purifying. B. The SDS-PAGE anlysis of Bacillus subtilis with pHT254 in 24 h culture solution supernatant and the prufied product. It was found that the experimental groups (the unpurified culture and purified product) had a significant band at 17 kDa, which was different from the expected band of about 34 kDa. M: Prestained Protein Ladder; Ct: the control group; UPC and PC: the unpurified and purified culture.

**Learn**

Upon closer examination of the circuit, an error was found in the initial vector: the Splip gene sequence that should have been on the 5 'end of the coding sequence(CDS) was mistakenly placed on the 3' end, resulting in Splip not being functionally active in the vector. The unknown 17 kDa protein obtained by nickel ion affinity chromatography may be the native exoprotein of pHT254 plasmid.

### The Cycle 2

**Design**

In order to solve this problem, the expression vector of the verification circuit is redesigned so that the components in it are theoretically functional. In this process, the new circuit takes special care that, in order to avoid the polyhistidine tag being incorrectly excised together with Splip, its sequence is placed on the 3 'end of the CDS.

![](https://static.igem.wiki/teams/5335/aspf2-splip/novel-guys.png)

**Figure 39. The plasmids constructed to express the Splip-Nlp20 and Splip-VDAL in *B.subtilis*.**

Each of the Splip-Nlp20 and Splip-VDAL is linked with a 6\*HIs-tag which is used for the future nickel ion affinity chromatography purifying.

**Build**

The new circuit was constructed in the same method as the initial. The preliminary confirmation of the success of the transformation of the vector (**Figure 40.**) was achieved through the polymerase chain reaction (PCR) of the transformed *E.coli* DH5α colony. Subsequently, the target *B. subtilis* 168 colony was identified through the aforementioned method. The positive band in E. coli DH5α is 1512 bp, which is 1075 bp in *B. subtilis* 168.

![](https://static.igem.wiki/teams/5335/aspf2-splip/bac-p.png)
**Figure 40. Agarose gel electrophoresis validation of PCR results of *E.coli* DH5α and *B.subtilis* 168 colonies.**

The first lane was loaded with DL2000 DNA ladder.Sizes were marked on the image. We chose 2× Magic Green Taq SuperMix.It contains DNA Polymerase, dNTP, and an loading buffer system, which allows amplification by adding only primers and templates,the product can be electrophoretic directly after PCR,simplify the experimental process and improve the repeatability of the results. The PCR reaction consisted of 15 μL 2 × Magic Green Taq SuperMix, 1.5 μL forward primer (3.75 mM), 1.5 μL reverse primer (3.75 mM), 11 μL H2O and 1 μL colony. A. M: DL2,000 DNA Marker; 1-6: Colony PCR bands for E.coli DH5α. The target bands are between 2000bp and 1000 bp; B. M: DL10,000 DNA Marker; 1-6: Colony PCR bands for B.subtilis 168. The target bands are approximately in a line with 1000 bp, the 5 and 6 bands with wrong position could be caused by too much sample volume.

**Test**

In the process of purifying the supernatant of engineered Bacillus subtilis culture liquid with new carrier, the concentration of target protein was not purified in the supernatant due to the shortage of experimental conditions such as bacterial liquid volume and culture time. In view of this, a SDS-PAGE analysis of the total protein of the bacterium body was performed to determine the expression of plant immune activators in Bacillus subtilis. Due to the unsatisfactory effect of conventional cracking methods on Bacillus subtilis, a set of lyase-ultrasonic crushing and alkali cracking methods were developed and showed good results in subsequent experiments. The expression of Splip-VDAL was verified by SDS-PAGE analysis of whole protein, but Splip-Nlp20 was too small to be determined on SDS-PAGE.

![](https://static.igem.wiki/teams/5335/aspf2-splip/3.png)
**Figure 41. The SDS-PAGE analysis of whole proteins of non-engineered *B.subtilis* 168 and *B.subtilis* 168 bacteriophage carrying expression vectors.**

M: Prestained Protein Ladder; WT: the non-engineered of *B.subtilis* 168; NV-1 and NV-2: the 2 loaded samples from the *B.subtilis* 168 containing the express vector. The target bands are between 35 kDa and 55 kDa, which are highlighted with black arrows.

**Learn**

The new circuit demonstrated the successful expression of Spip-fusion protein in Bacillus subtilis. However, due to the disadvantages of low secretion efficiency, low amount of bacterial solution used and short culture time, the verification of secretion labels needs to be completed. In the subsequent verification, the purification method of the target protein in the supernatant needs to be improved, and the purification efficiency can be further improved by salting-out - superpermeability - affinity chromatography.

### The Cycle 3

**Design**

Since no active VDAL protein was purified in the culture supernatant, a validation circuit containing VDAL-6\*His under the control of the lactose operon and T7 promoter was constructed and transformed into *E.coli* BL21 (DE3) for expression.

The constructed plasmid vector is illustrated in **Figure 42**.

![](https://static.igem.wiki/teams/5335/plant-cpps/18.png)

**Figure 42. Plasmid Vector**

**Build**

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 43**.

![](https://static.igem.wiki/teams/5335/plant-cpps/19.png)

**Figure 43. Agarose gel electrophoresis image of colony PCR products. (target band at 553 bp)**

**Test**

For functional validation, VDAL-6\*His was purified by nickel ion affinity chromatography. The enhanced effect of plant ETI was verified by ROS staining after incubation of purified VDAL-6\*His with Arabidopsis thanlia leaves to apparent ROS content changes (**Figure 44.**).

![](https://static.igem.wiki/teams/5335/plant-cpps/22.png)

**Figure 44. DAB staining images of Arabidopsis leaves.**

Treated with: A. ddH₂O (control), B. salicylic acid (SA) (100 μM), C. VDAL-6\*His protein (50 μg/mL), and D. a combination of SA (100 μM) and VDAL-6\*His protein (50 μg/mL).

**Learn**

In this cycle, the PTI promoting activity of VDAL was validated. These results provide strong evidence for the feasibility of the plants immunity enhancement module.

Nematode killing module

## The part of Cry6Aa2

## Cycle 1

### Design

Cry6Aa2 is our primary functional protein for killing nematodes, so we first demonstrated that it could be expressed. We designed to add a histidine tag to Cry6Aa2 protein to facilitate the purification of Cry6Aa2, and control the expression of Cry6Aa2 by adding lactose operon.[(BBa\_K5335006)](https://parts.igem.org/Part%3ABBa_K5335006)

### Build

We constructed the pET28a plasmid with lactose operon with Cry6Aa2 labeled with 6\*His, and introduced the constructed plasmid into *E.coli* BL21(DE3).

![](https://static.igem.wiki/teams/5335/cry6aa2/liucheng.png)
 **Figure 45. Flow chart of the construction of the expression carrier.**

Agarose gel electrophoresis of colony PCR products showed that we successfully constructed the expression vector.

![](https://static.igem.wiki/teams/5335/cry6aa2/clony-pcr.png)
 **Figure 46. M: DL2000 DNA Marker; 1-6: Colony PCR bands for *E.coli*.**

### Test

In the induced expression experiment, *E.coli* BL21(DE3) was induced by 1 M IPTG when OD600= reached 1.0, and further cultured for 8 hours. Subsequently, the bacteria were collected and cleaved, and the total protein extract was purified. The results of SDS-PAGE and Western Blot analysis showed that Cry6Aa2 protein was successfully expressed.

![](https://static.igem.wiki/teams/5335/cry6aa2/zutu2.png)
 **Figure 47. A:SDS-PAGE of purified sampleB:Western Blot of purified sample.**
M:Protein Marker.A:1 and 3 were Uninduced group, and 2 and 4 were Induction group.B:1 was Uninduced group,2 was Induction group.

### Learn

Although Cry6Aa2 protein was successfully expressed, a small amount of Cry6Aa2 protein was also expressed in the uninduced group, which we speculated was caused by low-dose leakage of lactose operon.

## Cycle 2

### Design

In order to enhance the permeability of Cry6Aa2 protein to nematodes, we fused cell penetrating peptides (CPPs) at its C-terminal and designed a fusion protein to connect Cry6Aa2, CPPs, and SpyCatcher through the (GGGGS)4 flexible linkage sequence. The design aims to ensure that the fusion protein can effectively bind to the SpyTag on the VLP surface after expression, achieve intracellular transfer of Cry6Aa2 protein through CPPs, and display it stably on the VLP surface.

### Build

We used a two-step recombination strategy to construct the expression vector. First, SpyTag-6\*His was recombined into the pET28a plasmid. In order to improve the efficiency of homologous recombination, this segment contained egfp gene as an unrelated sequence and formed a larger fragment with SpyTag-6\*His. Subsequently, the SpyCatcher-Cry6Aa2-CPPs fusion fragment (linked by the (GGGGS)4 flexible linkage sequence) was recombined into the pET28a plasmid that already contained the fragment SpyTag-6\*His.

![](https://static.igem.wiki/teams/5335/cry6aa2/recombine.png)
 **Figure 48. A. First recombination B. Second recombination.**

### Test

The transformed recombinant *E. coli* BL21(DE3) was cultured at 37°C for 12 hours, then the bacteria were collected and ultrasonically lysed. Then, the total protein extract was purified, and the obtained total protein and purified sample were analyzed by SDS-PAGE.SDS-PAGE showed that the complex protein was successfully expressed

![](https://static.igem.wiki/teams/5335/cry6aa2/222sp.png)
 **Figure 49. A:SDS-PAGE of total protein B:SDS-PAGE of purified sample.**
The weight of the target protein is 68.3 kDa.Ctrl:Empty *E.coli* BL21 (DE3). Ex. *E.coli* BL21 (DE3) that has been introduced into the expression vector.1 and 2 were purified samples.

### Learn

Non-specific bands were observed in SDS-PAGE analysis, indicating the presence of heteroproteins during purification. In the future, the purification conditions can be optimized to improve the purity of the target protein.

## shRNA that target key genes in nematode

### Design

In order to effectively inhibit the infection of nematodes on plants, we designed shRNA targeting the key genes 16D10 and flp. The parasitic peptide encoded by 16D10 gene interacts with plant transcription factors to promote nematode infection, while flp gene is closely related to the motor function of nematode.

![](https://static.igem.wiki/teams/5335/cry6aa2/seq-shrna.png)
 **Figure 50. The assembled RNA sequence.**

### Build

Both 16D10-shRNA and flp-shRNA are attached to an ms2-loading sequence with a stem-loop structure for assembly into VLP (virus-like particles).

![](https://static.igem.wiki/teams/5335/cry6aa2/stru-shrna.png)
 **Figure 51. Secondary structure of RNA.**

A:RNA containing 16D10 shRNA B:RNA containing flp shRNA

### Learn

Since flp gene plays no significant role in nematode infection of plants, we decided to retain only shRNA against 16D10 gene to inhibit nematode infection of plants.

Colonization

### Design

At first, through literature, we learned that Bacillus mainly relies on the assembly of TasA into amyloid fibrillary structures to form biofilms, and 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.
Therefore, We designed to combine TapA genes with the strong constitutive promoters p43 and SpoVG RBS , in order to increase the expression of TapA in Bacillus and enhance the colonization ability of Bacillus.

### Build

We constructed the gene circuit for high TapA expression and bound it to the shuttle plasmid pHT-254, and finally it is transferred into *B.subtilis* 168 by electrical transformation.

![](https://static.igem.wiki/teams/5335/holin-pghs/10-1-nignt-update/tapa-1.png)
 **Figure 52. TapA circuit.**

![](https://static.igem.wiki/teams/5335/colonization/pht254-tapa.png)
 **Figure 53. pHT254-tapA.**

### Test

By incubating GT(l) and untransformed *B.subtilis* 168 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 .

![](https://static.igem.wiki/teams/5335/colonization/bx-1.png)
 **Figure 54. Results of dissolving crystal violet A590 in different groups.**

GT(l) is the engineered bacterium that contains only TapA, Control is the blank control without adding bacteria.

The results showed that increasing the expression level of TapA could improve the biofilm generation ability of *B.subtilis* 168, but the difference is not very significant and does not meet our expectations.

### Learn

Through the analysis of the results, we believe that only increasing the TapA expression level is not enough to significantly improve the colonization ability of engineered bacteria.

### Redesign

Through further review of relevant literature, we found that Bacillus while also promoting biofilm formation by producing major and minor wall teichoic acid (WTA), and 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.
Therefore, we decided to add the GgaA gene to the previous gene circuit to see if we could further improve the ability of the engineered bacteria to colonize.

### Build

We constructed the gene circuit for high TapA and GgaA expression and bound it to the shuttle plasmid pHT-254, and finally it is transferred into *B.subtilis* 168 by electrical transformation.

![](https://static.igem.wiki/teams/5335/holin-pghs/10-1-nignt-update/cc-2.png)
 **Figure 55. TapA-GgaA circuit.**

![](https://static.igem.wiki/teams/5335/colonization/pht254-ggaa-tapa-1.png)
 **Figure 56. pHT254-tapA-GgaA.**

### Test

By incubating GT(l), GT(h) and untransformed *B.subtilis* 168 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 .

![](https://static.igem.wiki/teams/5335/colonization/bx2.png)

**Figure 57. Results of dissolving crystal violet A590 in different groups.**

GT(l) is the engineered bacterium that contains only TapA, GT(h) is the engineered bacterium that contains both GgaA and TapA, Control is the blank control without adding bacteria.

This time, the results not only showed that increasing the expression level of TapA could improve the biofilm generation ability of *B.subtilis* 168, but also showed that this effect was more significant after increasing the expression level of GgaA and TapA at the same time.

### Learn

Through the analysis of the results, we believe that the combination of different same functional modules can be superimposed to improve the purpose function. Unfortunately, due to time, we could not test the effect of only increasing GgaA expression. However, we hope that this study can provide research ideas for subsequent relevant studies.

Lysis & Release Module

### Design

Our engineered bacteria express VLP particles, and in order to release these particles outside the bacteria, we designed a circuit for lytic bacteria, which activates the lytic circuit by inducing expression, and completes the lytic bacteria and product release.

### Build

We chose two proteins, holin and peptidoglycan hydrolase. They are both naturally occurring proteins in Phage VB\_SUP-GOE1. The former can perforate the cell membrane of Bacillus subtilis, and the latter can hydrolyze peptidoglycan. We hope that the two can work together to trigger the lysis of the bacteria.

We bind them to a xylose operon bearing pHT-315 plasmid in order to control their expression and varified their function.

![](https://static.igem.wiki/teams/5335/holin-pghs/plasmid-map-hp.png)
 **Figure 58. Plasmid profile.**
![](https://static.igem.wiki/teams/5335/holin-pghs/hp-sequencing-test.png)
 **Figure 59. Sequencing result.**

### Test

Our lysis circuit is expected to work in *B.subtilis*, but we don't know as much about this bacterium as we do about E. coli. Initially, we planned to characterize the effects of bacterial lysis by measuring the growth curve. After searching the literature, we determined a suitable D-xylose induction concentration: 60 mM/L, and conducted multiple groups of pre-experiments, starting with the addition of D-xylose induction when the OD600 value of the bacteria solution was:0.1, 0.3, 0.5 and 0.8, respectively. However, in these pre-experiments, After several hours of continuous cultivation,the OD600 value of the experimental group and the control group was small different.

![](https://static.igem.wiki/teams/5335/holin-pghs/d-xylose-induced-lysis-formal-experiment1.png)
 **Figure 60.** The bacterial solution of the same recombinant strain was divided into two parts, one part were normal B.subtilis168 (Control),another part were transformed B.subtilis168 (Treatment).Then they were added with different concentrations of D-xylose as shown in the figure, sealed with a air-tight film, and cultured for 12 hours, after which the OD600 value was measured.

As can be seen from the figure, the OD600 value of recombinant bacteria under D-xylose induction was slightly lower than that of the group without D-xylose induction, which may indicate that our cleavage circuit played a role, but the effect was not obvious. We believe that this may be due to the rapid growth of Bacillus subtilis, and we suspect that xylose, as an energy substance that promotes the growth of Bacillus subtilis, will further narrow the gap between the experimental and control groups.

In order to further confirm the role of our circuit and verify these two conjectures, we conducted more pre-experiments. In view of the fact that Bacillus subtilis is an aerobic bacterium, we cultured Bacillus subtilis in a 10 mL bacterial vial, induced by adding D-xylose, and sealed the bacterial vial with an airtight membrane to create an anoxic environment.

After the success of the preliminary experiment, we designed a formal experiment in which we not only treated the recombinant Bacillus subtilis, but also added the non-recombinant Bacillus subtilis as a control.

![](https://static.igem.wiki/teams/5335/holin-pghs/d-xylose-induced-lysis-formal-experiment-hypoxia-1.png)
**Figure 61.** The bacterial solution of the same recombinant strain was divided into two parts, one part were normal B.subtilis168 (Control),another part were transformed B.subtilis168 (Treatment).Then they were added with different concentrations of D-xylose as shown in the figure, sealed with a air-tight film, and cultured for 12 hours, after which the OD600 value was measured.

The results showed that the OD600 value of recombinant bacteria was lower than that of non-induced group under different concentrations of D-xylose. The OD600 value of induced recombinant bacteria was lower than that of non-recombinant bacteria except 180 mM/L group. From this data, we can say that the lysis circuit does work. Moreover, the higher the concentration of D-xylose induced, the higher the OD600 value of recombinant bacteria, which also confirmed our conjusions to a certain extent.

### Learn

The whole process of constructing and characterizing the lysis curcit is not easy. Our lysis circuit is expected to work in *B.subtilis*, but we don't know as much about this bacterium as we do about E. coli.

By reviewing the literature and continuing pre-experiments to determine the best experimental operation, we have successfully characterized the cleavage circuit and obtained ideal experimental results. At the same time, a series of our guesses can also be confirmed to a certain extent, such as xylose as an energy material, the promotion of the growth of Bacillus subtilis will weaken the significance of our experimental results.

Suicide

### Design

Our engineered strain is an endophytic bacterium that colonizes and reproduces in the plant. Considering its high efficiency and portability, we chose light as a suicide method. We began to review and screen phototoxic proteins to select the best fit for the biosafety module.

### Build

We ended up with KillerRed (BBa K1184000). According to the study, KillerRed is a red phototoxic protein that produces reactive oxygen species (ROS) under yellow-green light (540-585 nm). Given that plants are usually green, i.e. reflect light close to 540 nanometers (green), KillerRed is suitable as a suicide module for this project. We codon optimized KillerRed to make it suitable for our engineered bacteria.

![](https://static.igem.wiki/teams/5335/suicide/figure-n-plasmid-profile-2.png)

**Figure 62. Plasmid profile.**

The expression of KillerRed is controlled by xylose operon and is induced by xylose.

### Test

We tested the suicide performance of KillerRed in engineered bacteria, which was manifested by poor sensitivity to different colors of light and suicide performance under artificial green light and sunlight. We found that KillerRed has a very obvious killing activity in engineered bacteria, and this effect is mainly caused by green light excitation. Detailed results can be found in Result.

![](https://static.igem.wiki/teams/5335/suicide/1-data-4.png)

**Figure 63. 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.

![](https://static.igem.wiki/teams/5335/suicide/1-data-1.png)

**Figure 64. 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.

### Learn

In the actual testing process, we found that the selection of induction conditions had a strong influence on the results, indicating that the amount of KillerRed expressed in cells is highly correlated with the suicide effect, which is an important learning experience for the research and development project. Before implementation, high expression and stability of the selected protein should be ensured to ensure that it can be effectively tested and validated in experimental situations.

[ ]
Reference

[1] Liu H, Zhang L, Wang W, Hu H, Ouyang X, Xu P, Tang H. An Intelligent Synthetic Bacterium for Chronological Toxicant Detection, Biodegradation, and Its Subsequent Suicide. Adv Sci (Weinh). 2023 Nov;10(31):e2304318.

[2] Gilbert C, Howarth M, Harwood CR, Ellis T. Extracellular Self-Assembly of Functional and Tunable Protein Conjugates from Bacillus subtilis. ACS Synth Biol. 2017 Jun 16;6(6):957-967.

[3] Zhang F, Peng D, Cheng C, Zhou W, Ju S, Wan D, Yu Z, Shi J, Deng Y, Wang F, Ye X, Hu Z, Lin J, Ruan L, Sun M. Bacillus thuringiensis Crystal Protein Cry6Aa Triggers Caenorhabditis elegans Necrosis Pathway Mediated by Aspartic Protease (ASP-1). PLoS Pathog. 2016 Jan 21;12(1):e1005389.

[4] Biela AP, Naskalska A, Fatehi F, Twarock R, Heddle JG. Programmable polymorphism of a virus-like particle. Commun Mater. 2022 Feb 7;3:7.

[5] Naskalska A, Heddle JG. Virus-like particles derived from bacteriophage MS2 as antigen scaffolds and RNA protective shells. Nanomedicine (Lond). 2024;19(12):1103-1115.

[6] Jovanovic M, Lawton E, Schumacher J, Buck M. Interplay among Pseudomonas syringae HrpR, HrpS and HrpV proteins for regulation of the type III secretion system. FEMS Microbiol Lett. 2014 Jul;356(2):201-11.

[7] Ma RJ, Wang YH, Liu L, Bai LL, Ban R. Production enhancement of the extracellular lipase LipA in Bacillus subtilis: Effects of expression system and Sec pathway components. Protein Expr Purif. 2018 Feb;142:81-87.

[8] Jiang S, Zheng W, Li Z, Tan J, Wu M, Li X, Hong SB, Deng J, Zhu Z, Zang Y. Enhanced Resistance to Sclerotinia sclerotiorum in Brassica rapa by Activating Host Immunity through Exogenous Verticillium dahliae Aspf2-like Protein (VDAL) Treatment. Int J Mol Sci. 2022 Nov 12;23(22):13958.

[9] Böhm H, Albert I, Oome S, Raaymakers TM, Van den Ackerveken G, Nürnberger T. A conserved peptide pattern from a widespread microbial virulence factor triggers pattern-induced immunity in Arabidopsis. PLoS Pathog. 2014 Nov 6;10(11):e1004491.

[10]Soliman A, Laurie J, Bilichak A, Ziemienowicz A. Applications of CPPs in Genome Editing of Plants. Methods Mol Biol. 2022;2383:595-616.

[11]Chen YJ, Liu BR, Dai YH, Lee CY, Chan MH, Chen HH, Chiang HJ, Lee HJ. A gene delivery system for insect cells mediated by arginine-rich cell-penetrating peptides. Gene. 2012 Feb 10;493(2):201-10.

[12]Du C, Cao S, Shi X, Nie X, Zheng J, Deng Y, Ruan L, Peng D, Sun M. Genetic and Biochemical Characterization of a Gene Operon for trans-Aconitic Acid, a Novel Nematicide from Bacillus thuringiensis. J Biol Chem. 2017 Feb 24;292(8):3517-3530.

[13]Romero D, Vlamakis H, Losick R, Kolter R. Functional analysis of the accessory protein TapA in Bacillus subtilis amyloid fiber assembly. J Bacteriol. 2014 Apr;196(8):1505-13.

[14]Takemoto K, Matsuda T, Sakai N, Fu D, Noda M, Uchiyama S, Kotera I, Arai Y, Horiuchi M, Fukui K, Ayabe T, Inagaki F, Suzuki H, Nagai T. SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation. Sci Rep.

[15]Willms IM, Hertel R. Phage vB\_BsuP-Goe1: the smallest identified lytic phage of B.subtilis. FEMS Microbiol Lett. 2016 Oct;363(19):fnw208.

[16]Xu Z, Zhang H, Sun X, Liu Y, Yan W, Xun W, Shen Q, Zhang R. Bacillus velezensis Wall Teichoic Acids Are Required for Biofilm Formation and Root Colonization. Appl Environ Microbiol. 2019 Feb 20;85(5):e02116-18.

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](https://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

Results

![](https://static.igem.wiki/teams/5335/description/tree.png)

Salicylic Acid Biosensor
VLP Carrier
Plants Immunity Enhancement
Nematode Killing Module
Colonization
Lysis & Release
Suicide

Salicylic Acid Biosensor

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.

Virus-like particles delivery system expression and assembly

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.

![](https://static.igem.wiki/teams/5335/ms2-spy-e/self-assembly.png)
**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 Psal. Upstream of *ms2 cp*, we have reserved a place for access to the salicylic acid response element NahR.

![](https://static.igem.wiki/teams/5335/ms2-spy-e/ms2-plasmid.png)
 **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.

![](https://static.igem.wiki/teams/5335/ms2-spy-e/success-bacp-and-seq.png)
 **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.**

![](https://static.igem.wiki/teams/5335/ms2-spy-e/f4.png)
 **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.

![](https://static.igem.wiki/teams/5335/ms2-spy-e/f5.png)
 **Figure 9. SDS-PAGE and Western Blot of target protein.**
**a** and **b** are the eluents with the second highest protein concentration and the highest protein concentration in the purification process.
 **A.** SDS-PAGE of target protein. 1. Impurity protein eluent a. 2. Impurity protein eluent b. 3. Target protein eluent a.
4. BL21 (DE3) strain. 5. Supernatant. 6. Precipitate. 7. Target protein eluent b. MW: Molecular weight.
 **B.** Western Blot of target protein. 1. Target protein eluent a. 2. Target protein eluent b. MW: Molecular weight.

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.

![](https://static.igem.wiki/teams/5335/ms2-spy-e/new-agarose-pro-and-rna.png)
 **Figure 10. Gel retardation analysis different staining conditions.**
The left image is the result after dyeing with Safe Red dye, and the right image is the result after dyeing with G250 dye. The image on the left is obtained after image inversion by image processing software. Line 1: DNA loading buffer. Line 2: DNA loading buffer + TNE buffer. Line 3: Purified protein + TNE buffer + DNA/RNAase + DNA loading buffer. Line 4: DNA loading buffer + TNE buffer + DNA/RNAase

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.

![](https://static.igem.wiki/teams/5335/ms2-spy-e/f6.png)
 **Figure 11. Density gradient centrifugation results and correct assembly of VLP particles.**
**A.** Density gradient centrifugation results. The **red arrows** indicate the separated band.
**B,** **C.** Image of correctly assembled VLP under a transmission electron microscope. The **black arrows** represent proteins attached to the particle surface.

Plants Immunity Enhancement

## 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**.

![](https://static.igem.wiki/teams/5335/plant-cpps/41.png)

**Figure 12. Experimental circuit design diagram**

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**.

![](https://static.igem.wiki/teams/5335/plant-cpps/2.png)

**Figure 13. Plasmid Map**

#### 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**.

![](https://static.igem.wiki/teams/5335/plant-cpps/3.png)

**Figure 14. Agarose gel electrophoresis image of colony PCR products (target band at 2200 bp)**

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**.

![](https://static.igem.wiki/teams/5335/plant-cpps/4.png)

**Figure 15. SDS-PAGE gel electrophoresis image.**
Compared with the control, a target band of approximately 53.7 kDa was detected.

**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**.

![](https://static.igem.wiki/teams/5335/plant-cpps/5.png)

**Figure 16. SDS-PAGE gel electrophoresis image.**

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**.

![](https://static.igem.wiki/teams/5335/plant-cpps/6.png)

**Figure 17. Western Blot Image.**

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.

![](https://static.igem.wiki/teams/5335/aspf2-splip/novel-guys.png)
**Figure 18. The plasmids constructed to express the Splip-Nlp20 and Splip-VDAL in *B.subtilis*.**
 Each of the Splip-Nlp20 and Splip-VDAL is linked with a 6\*HIs-tag which is used for the future nickel ion affinity chromatography purifying.

#### 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.

![](https://static.igem.wiki/teams/5335/aspf2-splip/3.png)

**Figure 19. The SDS-PAGE analysis of whole proteins of non-engineered *B. subtilis*168 and *B. subtilis* bacteriophage carrying expression vectors.**
M: Prestained Protein Ladder; WT: the wild type of *B.subtilis* 168, with no genetic engineering; NV-1 and NV-2: the 2 loaded samples from the B.subtilis 168 containing the express vector. The target bands are between 35 kDa and 55 kDa, which are highlighted with black arrows.

### 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**. ![](https://static.igem.wiki/teams/5335/plant-cpps/42.png) **Figure 20. Experimental circuit design diagram** The constructed plasmid vector is illustrated in **Figure 21**. ![](https://static.igem.wiki/teams/5335/plant-cpps/18.png) **Figure 21. Plasmid Vector** 1.3.1 Experimental VerificationTransformation 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**. ![](https://static.igem.wiki/teams/5335/plant-cpps/19.png) **Figure 22. Agarose gel electrophoresis image of colony PCR products (target band at 553 bp)** 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**. ![](https://static.igem.wiki/teams/5335/plant-cpps/20.png) **Figure 23. SDS-PAGE gel electrophoresis image.** 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**. ![](https://static.igem.wiki/teams/5335/plant-cpps/21.png) **Figure 24. SDS-PAGE gel electrophoresis image.** The bands labeled 1, 2, 3, and 4 in the figure represent the elution fractions obtained sequentially using Elution Buffer. 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**. ![](https://static.igem.wiki/teams/5335/plant-cpps/26.png) **Figure 25. DAB staining images of Arabidopsis leaves.** Treated with: **A.** ddH₂O (control). **B.** salicylic acid (SA) (100 μM). **C.** SpyCatcher-VDAL-CPPs protein (50 μg/mL). **D.** a combination of SA (100 μM) and SpyCatcher-VDAL-CPPs protein (50 μg/mL).**E.** VDAL-6\*His protein (50 μg/mL). **F.** a combination of SA (100 μM) and VDAL-6\*His protein (50 μg/mL) . 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**. ![](https://static.igem.wiki/teams/5335/plant-cpps/27.png) **Figure 26. Inverted 8-bit grayscale images of Arabidopsis leaves following DAB staining.** Treated with: A. ddH₂O (control), B. salicylic acid (SA) (100 μM), C. SpyCatcher-VDAL-CPPs protein (50 μg/mL), D. a combination of SA (100 μM) and SpyCatcher-VDAL-CPPs protein (50 μg/mL),E. VDAL-6\*His protein (50 μg/mL), F. a combination of SA (100 μM) and VDAL-6\*His protein (50 μg/mL) . 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**. ![](https://static.igem.wiki/teams/5335/plant-cpps/28.png) **Figure 27. Images of DAB-based ROS Assay.** 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.** ![](https://static.igem.wiki/teams/5335/plant-cpps/43.png) **Figure 28. Experimental circuit design diagram** The constructed plasmid vector is illustrated in **Figure 29**. ![](https://static.igem.wiki/teams/5335/plant-cpps/11.png) **Figure 29. Plasmid Vector** 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**. ![](https://static.igem.wiki/teams/5335/plant-cpps/12.png) **Figure 30. Agarose gel electrophoresis image of colony PCR products (target band at 752 bp)** 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**. ![](https://static.igem.wiki/teams/5335/plant-cpps/16.png) **Figure 31.Supernatant of bacterial lysate and treated root tissue.** 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**. ![](https://static.igem.wiki/teams/5335/plant-cpps/14.png) **Figure 32. Confocal microscopy images of root tissues.** **A.** AmCyan fluorescence channel showing. **B.** No-stain control. **C.** Merged image of AmCyan fluorescence and brightfield. **D.** High-magnification view of root hair cells in the merged image. 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**. ![](https://static.igem.wiki/teams/5335/plant-cpps/15.png) **Figure 33. Confocal fluorescence microscopy images of protoplasts prepared from Arabidopsis roots. **A.** AmCyan fluorescence channel showing. **B.** No-stain control. **C.** Merged image of AmCyan fluorescence and brightfield.** 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.

Nematode Killing Module

### 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.

![](https://static.igem.wiki/teams/5335/cry6aa2/111.png)
 **Figure 34.Cry6Aa2-6\*His(pET28a)plasmid vector.**

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

![](https://static.igem.wiki/teams/5335/cry6aa2/clony-pcr.png)
 **Figure 35.Colony PCR gel electrophoresis results.**

M: DL2000 DNA Marker; 1-6: Colony PCR bands for *E. coli*BL21(DE3)

*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.

![](https://static.igem.wiki/teams/5335/cry6aa2/zutu2.png)
 **Figure 36.Cry6Aa2 protein purification results**
**A.** SDS-PAGE of pourified sample. **B.** Western Blot of purified sample.
M:Protein Marker.A:1 and 3 were Uninduced group, and 2 and 4 were Induction group.B:1 was Uninduced group,2 was Induction group.

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.

![](https://static.igem.wiki/teams/5335/cry6aa2/sp.png)

 **Figure 37.SpyCatcher-Cry6Aa2-CPPs+SpyTag-6\*His(pET28a)plasmid vector.**

*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.

![](https://static.igem.wiki/teams/5335/cry6aa2/222sp.png)
 **Figure 38.SDS-PAGE gel electrophoresis results**
**A.** SDS-PAGE of total protein. **B.** SDS-PAGE of purified sample
The weight of the target protein is 68.3 kDa.
**Ctrl:**Empty *E. coli* BL21 (DE3).**Ex:***E. coli* BL21 (DE3) that has been introduced into the expression vector.1 and 2 were purified samples.

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.

![](https://static.igem.wiki/teams/5335/tbr/hplc-result.png)
 **Figure 39. The HPLC detection pattern.**
**A.** 10 μM TAA standard. **B.** 100 μM TAA standard. **C.** Sample 1: Medium from single colony 1 fermented for 24h. **D.** Sample 2: Medium from a single colony 2 fermented for 24h.

### 16D10 shRNA design

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

![](https://static.igem.wiki/teams/5335/cry6aa2/16d-shrna.png)
 **Figure 40. Designed 16D10 shRNA sequence and structure.**

Colonization

*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.

![](https://static.igem.wiki/teams/5335/holin-pghs/10-1-nignt-update/ccc-1.png)
 **Figure 41.Two different circuits.**

![](https://static.igem.wiki/teams/5335/holin-pghs/10-1-nignt-update/pht254-tapa-n-1.png)
 **Figure 42.Construction of pHT254 Plasmid with tapA.**

![](https://static.igem.wiki/teams/5335/holin-pghs/10-1-nignt-update/pht254-ggaatapa-n-1.png)
 **Figure 43.Construction of pHT254 Plasmid with ggaA and tapA.**

Firstly, we successively transformed *E. coli*DH5α 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.

![](https://static.igem.wiki/teams/5335/colonization/dh5-pcr.png)

 **Figure 44 .Agarose gel electrophoresis validation of PCR results of *E.coli* DH5α colonies.**
M. DL5000 DNA Marker. 1-9.Nine single colonies obtained after transformation

Then we transferred the extracted plasmids into *B. subtilis*168 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. subtilis*168 **(Figure 45)**. As can be seen from the figure, GT(l) only expresses TapA, while GT(h) expresses both GgaA and TapA.

![](https://static.igem.wiki/teams/5335/colonization/sds.png)
 **Figure 45.SDS-PAGE analysis of GgaA and TapA expression.**
**GT(l).** Engineered bacteria that contains only TapA. **GT(h).** Engineered bacteria contains both GgaA and TapA. **Control.** the whole protein of *B. subtilis* 168.

Then, by incubating GT(l) and untransformed *B. subtilis*168 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)**.

![](https://static.igem.wiki/teams/5335/colonization/jjz-1.png)

 **Figure 46. Crystal violet staining of biofilm.**
**1-3:** is the result of GT(l) dyeing. **4-6:** is the result of WT dyeing.

![](https://static.igem.wiki/teams/5335/colonization/bx-1.png)
 **Figure 47. Results of dissolving crystal violet A590 in different groups.**
**GT(l).** Engineered bacteria that contains only TapA. **Control.** Blank control without adding bacteria.

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. subtilis*168.

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)**.

![](https://static.igem.wiki/teams/5335/colonization/jm-1.png)
 **Figure 48.Biofilm growth results.**
From top to bottom, they are GT(l), GT(h), and WT.
Due to the limitation of shooting equipment, only 3 pieces are shown in each group

![](https://static.igem.wiki/teams/5335/colonization/bx2.png)
 **Figure 49.Results of dissolving crystal violet A590 in different groups.**
**GT(l).** Engineered bacterium contains only TapA.**GT(h).** Engineered bacteria contains both GgaA and TapA. **Control.**blank control without adding bacteria.

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.

Lysis & Release

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.
![](https://static.igem.wiki/teams/5335/holin-pghs/circuit/hp-circuit-6.png)
**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 PhrpL.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).
![](https://static.igem.wiki/teams/5335/holin-pghs/circuit/xyl-hp-circuit-5.png)
**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.

![](https://static.igem.wiki/teams/5335/holin-pghs/plasmid-map-hp.png)
**Figure 52. Varified lysis circuit.**

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.

![](https://static.igem.wiki/teams/5335/holin-pghs/agarose-gel-electrophoresis-validation-of-pcr-results-of-b-subtilis168-colonies.png)

 **Figure 53. Agarose gel electrophoresis validation of PCR results of *B.subtilis* 168 colonies.**
**M:** DL1000 DNA Marker; 1-6: Six transformed *B. subtilis* 168 single colonies .

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.

![](https://static.igem.wiki/teams/5335/holin-pghs/d-xylose-induced-lysis-formal-experiment1.png)
 **Figure 54. Bacterial OD600 over time.**

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.

![](https://static.igem.wiki/teams/5335/holin-pghs/d-xylose-induced-lysis-formal-experiment-hypoxia-1.png)
 **Figure 55.Bacterial OD600 over time (hypoxia).**

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.

Suicide

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.

![](https://static.igem.wiki/teams/5335/safety/light-sensitive-suicide-cricuit.jpg)

 **Figure 56. Light-sensitive suicide cricuit.**

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. subtilis*168 by electrical transformation. The part has been verified by sequencing.

![](https://static.igem.wiki/teams/5335/suicide/figure-n-plasmid-profile-2.png)

 **Figure 57. Plasmid profile.**
The expression of KillerRed is controlled by xylose operon and is induced by xylose.

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.

![](https://static.igem.wiki/teams/5335/suicide/figure-n-phenotypic-result-comparison-chart.jpeg )

 **Figure 58. Phenotypic result comparison chart.**
The left is the blank group, the right is the induction group, the liquid color of the obvious induction group turned red.

![](https://static.igem.wiki/teams/5335/suicide/1-data-3.png)
 **Figure 59. The chang of bacterial culture OD600 over illumination time.**

**Control group:** Treated with sterile water.**Experimental group:**Treated with xylose concentrated solution, followed by light for a total of 2 hours.

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.
![](https://static.igem.wiki/teams/5335/suicide/1-data-4.png)
 **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:

![](https://static.igem.wiki/teams/5335/suicide/1-data-1.png)
 **Figure 61. The chang of bacterial culture OD600 over illumination time.**

**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.