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].

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.

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].

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].

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].

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].

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.

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] .

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.

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.

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.

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.

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

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).
Figure 28. First homologous recombination.
Two recombinations are planned to complete the construction.
Figure 29. Colony PCR for fragments containing spycatcher-egfp.
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.
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 P_sal 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.) .
Figure 32. Reconstructed plasmid vector.
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.).
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.
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.

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.

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.

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.).

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.

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.

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.

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.

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.

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.).

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.

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)

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).

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

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.

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.

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

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.

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).

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.

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.

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 .

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.

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 .

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.

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.

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.

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.

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.

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.

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.

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.