RESULTS

We constructed plasmids capable of expressing Cry5Ba and Cry6Aa as well as their fusion proteins, and verified the successful construction and transformation of the plasmids.

Analysis: The PCR amplification bands of Cry5BA (approximately 1800 bp), Cry6Aa (approximately 1200 bp), the fusion protein (approximately 3000 bp), and the pET-28a vector (approximately 5000 bp) align with the theoretical lengths estimated based on the designed primer positions, demonstrating that these target genes have been successfully obtained.

Analysis: From the result of plate coating of LB solid medium with antibiotic Kan, the medium successfully grew dispersed single colonies, which indicated that the bacterium was successfully screened and transformed.

Analysis: The sequencing result can show that the target element and the plasmid vector were correctly connected; there were no heterogeneous peaks in the sequencing result, and the sequence of the actual plasmid is identical with the designed plasmid.

Analysis: The protein bands on the western blot gel for Cry5Ba (approximately 65.45 kDa), Cry6Aa (approximately 44.33 kDa), and the fusion protein (approximately 110 kDa) corresponded to their theoretical lengths based on their molecular weights, indicating that these target proteins were successfully expressed in the engineered bacteria.

Analysis: According to the survival curve, Cry6Aa has already begun to cause the death of nematodes, while the killing effect of Cry5Ba is not obvious. At 96 hours, due to the poor effect of the FUDR we used, a large number of larvae appeared, so we terminated this experiment.

Analysis: To accelerate the insecticidal rate, in the second set of survival analysis experiments, we modified the original experimental conditions by directly dropping protein solutions onto the culture medium. The results showed that 24 hours after protein application, the survival rates of the Cry5Ba group, Cry6Aa group, and Fusion protein group were all lower than 6%, whereas the survival rate of the control group remained higher than 75%. These findings confirm that both Cry5Ba protein and Cry6Aa protein, as well as their fusion protein, exhibit excellent insecticidal efficacy, which is consistent with our initial hypothesis.

We aim to design the E. coli to be a factory that can turn L-Tyr and malonate into naringenin. We transformed three plasmids containing six genes into the same E. coli. Then we conducted verification experiments on E.coli to ensure the enzymes were correctly expressed and functioning well.

The three plasmids containing the target genes were synthesized by company. Then we transformed the plasmids into the E. coli.

We used the method of electric transformation to simultaneously transform two plasmids into the E. coli. And then we coated the transformed E. coli on board that has two kinds of corresponding antibiotics to see if the transformation was successful.

Analysis: The transformed E. coli can grow on the board that has two kinds of corresponding antibiotics---Amp and Cap. This indicates that the transformed E. coli has the resistance genes of these two antibiotics, which is to say that the transformation process was likely successful.

We made the former E. coli into chemically competent cells and then we used the method of chemical transformation to transform the third plasmid into the E. coli in order to raise the successful rate of obtaining engineering bacteria that has three plasmids. Then we coated the transformed E. coli on the board that has three kinds of corresponding antibiotics---Amp、Smr and Cap.

Analysis: The transformed E. coli can grow on the board that has three kinds of corresponding antibiotics---Amp、 Smr and Cap. This indicates that the transformed E. coli has the resistance genes of these three antibiotics, which is to say that the transformation process was likely successful. Then we sent the board for company to conduct bacteria PCR. The result shows that the engineering bacteria has the three constructed plasmids(the six targeted genes), which is to say that the transformation processes in parts 1.1 and 1.2 were successful.

In our experiment, it was essential to determine whether E. coli successfully expressed the target enzyme we needed. Therefore, we used SDS-PAGE for verification and investigated whether temperature and the addition of an inducer are factors affecting the expression of E. coli.

We designed the experiment as follows: the control group consisted of engineered E. coli cultured at 16°C, 25°C, and 37°C without the inducer, while the experimental group comprised engineered E. coli cultured at the same three temperatures (16°C, 25°C, 37°C) with the inducer added. After processing, the samples were loaded into the gel lanes, and the results of the protein gel were observed.

Analysis:For the control group, the results showed that almost no target protein was detected in E. coli cultured at 37°C, whereas clear target protein bands were visible in the samples cultured at 16°C and 25°C. This indicates that even without inducing the engineered E. coli, we could still obtain the desired target enzyme within this specific temperature range. For the experimental group, the protein band of E. coli cultured at 37°C was the faintest, while the band intensities of the samples cultured at 16°C and 25°C were nearly identical. Meanwhile, due to the distortion of the gel, the position of the matC band may not be accurate.But in combination with the sequencing results, the result can still suggest that the expression levels of E. coli were approximately the same within this temperature range.(16℃ and 25℃)

Although the lack of a His-tag prevented us from performing Western Blotting, we still obtained the desired results: the engineered E. coli strain successfully expressed the target protein we needed, and this expression could be achieved without induction within a specific temperature range (indicating that the gene we introduced has a strong expression capacity).

The successful construction of the naringenin biosynthetic pathway in the engineered Escherichia coli required a reliable method to confirm the production of this target intermediate. Since naringenin contains a conjugated ring system with phenolic hydroxyl groups, it exhibits strong and characteristic ultraviolet (UV) absorption. Literature indicates a definitive absorption maximum for naringenin at 285 nm. Therefore, we established a High-Performance Liquid Chromatography (HPLC) method with UV detection specifically tuned to this wavelength. This approach was designed to unambiguously identify and quantify naringenin synthesis by the engineered E. coli strain based on its intrinsic photophysical properties.

Analysis: Based on the chromatographic results, the detection method was validated using standard compounds, confirming its reliability for our analytical purposes. Comparative analysis revealed that the sample containing the engineered E. coli strain exhibited a peak at 7.1 minutes, which aligns with the retention time of the naringenin standard. Moreover, no corresponding peak was observed in the blank control. These results indicate that the engineered E. coli successfully produced naringenin, demonstrating the effective construction of the intended biosynthetic pathway.

We aim to design the B. subtilis to be a factory that can turn naringenin into luteolin. We transformed one plasmid containing two genes into the B. subtilis. Then we conducted verification experiments on B. subtilis to ensure the enzymes were correctly expressed and functioning well.

First we constructed the plasmid containing the two targeted genes. Then we transformed it into the B. subtilis.

We used the method of PCR to copy fragments of genes and vector, and then we used the method of homologous recombination to combine the fragments. Then we transformed the plasmid into the B. subtilis as Experiment group and coated it on the board that has the corresponding antibiotic---Kan to see if the transformation process was successful. Simultaneously, we transformed an empty pBE2R plasmid into the Bacillus subtilis as Control group. In this part, we attempted to construct pBE2R plasmid for two times. But we failed for the first time because the homology of the fragments was poor. ( Because we have many similar sequences to combine ) Then we redesigned the pBE2R plasmid and deleted one promoter and one terminator to increase homology and then we tried the second time.

Analysis: The transformed B. subtilis can grow on the board that has Kan. This indicates that the transformed B. subtilis has the resistance gene of Kan, which is to say that the transformation process was likely successful.

Analysis: This is the bacteria PCR result of pBE2R. The fragment frag1’s length is 1200. Although the length of the band shown in the result is not exactly 1200bp, but based on the sequence result and the fact that the band is a result of bacteria PCR, we can reckon that the transformation process was successful.

Analysis: The sequencing result shows that the sequence we designed was the same as the measured sequence. This indicates that the transformation process was successful.

Whether B. subtilis successfully expresses the target enzyme determines our ability to convert naringenin to luteolin. Therefore, it is particularly important to confirm whether the protease expressed by the engineered B. subtilis is the target protein.

To obtain more accurate results during plasmid construction, we added a His-tag to the plasmid introduced into Bacillus subtilis. This allowed the expressed product to be specifically recognized by antibodies, enabling us to obtain clear images and accurate results via Western Blotting.

We conducted preliminary verification using SDS-PAGE.

To verify whether the temperature can affect the expression of the target protein, we redesigned the experiment:

Control group: B. subtilis transformed with the empty plasmid (i.e., pBE2R) and cultured at 16°C, 25°C, and 37°C.

Experimental group: B. subtilis transformed with the normal plasmid (i.e., pBE2R-FNS-F3’H) and cultured at the same three temperatures (16°C, 25°C, 37°C).

Analysis:The experimental results showed two key findings:

Temperature had little effect on the expression of the engineered B. subtilis—the B. subtilis transformed with the normal plasmid exhibited expression at all three temperatures, with bands of similar intensity.

Unexpectedly, B. subtilis transformed with the empty plasmid also showed a relevant band in the target molecular weight range. Under normal circumstances, transformed with an empty plasmid should not enable Bacillus subtilis to produce the target product. The appearance of this band might be attributed to one of the following two reasons:

1.Experimental operation error, where the control group was actually B. subtilis transformed with the normal plasmid.

2.The obtained expression product was not the target protein we needed.

Thus, we decided to use Western Blotting to verify whether the obtained protein was the target protein and to clarify the reason for the band appearance in the control group.

In the previous experiment, we set up groups with different temperatures, but the gel results showed that both B. subtilis transformed with the normal plasmid and those transformed with the empty plasmid exhibited target bands.

Analysis:Subsequent Western Blotting of this protein gel further confirmed that all these bands corresponded to the target protein.

This ruled out the possibility that the obtained protein was not the desired target protein, leading us to suspect that either the B. subtilis in the control group was also transfected with the normal plasmid or there was an error during sample loading onto the protein gel.

To resolve this, we redesigned the experiment:

Control group: B. subtilis transformed with the empty plasmid (i.e., pBE2R) and cultured at 25°C.

Experimental group: B. subtilis transformed with the normal plasmid (i.e., pBE2R-FNS-F3’H) and cultured at 25°C.

Analysis:After gel electrophoresis, Western Blotting was performed on the obtained protein gel. The images clearly showed that the B. subtilis transformed with the normal plasmid exhibited a target band, while no target band was observed in the B. subtilis transformed with the empty plasmid.

This successfully confirmed that the earlier appearance of a band in the B. subtilis transformed with the empty plasmid was due to experimental operation errors, and verified that we had obtained the desired target product.

For the engineered B. subtilis, which was designed to convert naringenin into the final product luteolin, the analytical method needed to detect and distinguish both compounds simultaneously. Luteolin, sharing a similar flavonoid structure but with extended conjugation, possesses a distinct UV absorption profile, with a reported maximum at 350 nm. To monitor the intended bioconversion, we developed a unified HPLC-UV method capable of detecting both naringenin (at 285 nm) and luteolin (at 350 nm) within a single run. This dual-wavelength detection strategy was essential to verify the specific activity of the B. subtilis strain by confirming not only the presence of luteolin but also the concomitant consumption of the naringenin precursor.

Analysis: In contrast, the sample with the engineered B. subtilis strain did not show any peak at the retention time corresponding to the luteolin standard, while the blank control again remained clear of such signals. This suggests that the conversion from naringenin to luteolin was not achieved, indicating a failure in the functional assembly of the pathway in the engineered B. subtilis.

We planed to screen phages targeting the B. subtilis BS168 strain from the environment to achieve regulation of engineered bacteria. After that, we aim to construct a plasmid containing “lsl” component and transform it into the E. coli to verify its function. While eventually in the application process, we will use phages as a medium to import this molecular switch.

We constructed and transformed a plasmid containing CHS and CHI genes and “lsl” component and a plasmid containing cre gene into the same E. coli as Experiment group. We tried for two times but we failed for the first time probably because the effect of the “cre” gene was too strong causing the lac operator to break. And we transformed a plasmid containing CHS and CHI genes and “lsl” component and an empty plasmid into the same E. coli as Control group.

Analysis: The transformation result of the Control group and the Experiment group shows that the transformed Escherichia coli(E.coli) can grow on the board that has two corresponding antibiotics. This indicates that the transformation process was likely successful.

Analysis: The sequencing result shows that there is a blank in the blue rectangular area, which is the position of the “lsl” component. This indicates that the transformation process was successful and cre gene was functioning well.

Analysis: The sequence result shows that the sequence we designed was the same as the measured sequence. This indicates that our construction of pCDF-cre plasmid using the method of PCR was successful.

To confirm whether our Cre-loxP system functions properly, we decided to conduct verification experiments in Escherichia coli (E. coli) BL21. The experiment was designed as follows: E. coli transfected with the pCDFDuet-CHS-lsl-CHI plasmid served as the control group, while E. coli co-transfected with the pCDFDuet-CHS-lsl-CHI plasmid and pCDFDuet-Cre plasmid served as the experimental group. Our hypothesis was that if the constructed Cre-loxP system was correct, the control group would be unable to express the target protein, whereas the experimental group would successfully express it.

Protein samples obtained from the above experiment were analyzed using SDS-PAGE, and the gel image results were as follows:

Analysis:First, a band corresponding to CHS was observed in both the control group and the experimental group, indicating that the plasmids were successfully transfected into E. coli and induced for protein expression. However, a band corresponding to the CHI protein was detected in both groups, suggesting that CHI was expressed in E. coli of both the control and experimental groups.

To identify the cause of the CHI band appearance, we performed Western Blot analysis on the protein gel.

Analysis: In the Western Blot image, CHI bands were still detected in both groups of E. coli (note: only CHI carries a His-tag).

Based on the above results, we proposed the following potential reasons:

1.The pCDFDuet-CHS-lsl-CHI plasmid transfected into the control group was contaminated with Cre. This contamination would lead to the excision of the terminator by Cre (a band suspected to correspond to Cre was observed at the respective position in the control group’s protein gel).

2.The terminator in the plasmid failed to exert its intended function.

In future experiments, we will further investigate the cause of the CHI band appearance in the control group to verify whether our Cre-loxP system indeed functions as expected.

We collected soil and water samples from flower beds and lakes across the campus of Huazhong University of Science and Technology. Each sample was co-incubated with bacterial suspensions of either Escherichia coli(E.coil) BL21 strain or Bacillus subtilis BS168 strain for amplification. Phages were subsequently screened using the double agar overlay assay and purified via the phage streaking method.

Analysis: Figure 23 shows the plaque obtained for the phages targeting engineered bacteria. The plaques were characterized by a transparent center with cloudy edges. The results in Figure 23 confirm the successful isolation of phages from environmental soil samples that are specific to the two engineered host bacteria. Evidence of their bactericidal efficacy is provided by the formation of clear plaques in double-layer agar assays. At the same time, we found that even for the phage plaques targeting the same engineered bacteria, they present different sizes and different degrees of roughness at the edges (Fig 23a & Fig 23b; Fig 23c & Fig 23f), which also indicates that the phages we isolated are not a single species but a mixed group. It is proposed that this heterogeneous phage community collectively contributes to the overall antibacterial outcome.

Analysis: TEM analysis reveals a diversity of phage morphologies in the E. coli phage-enriched liquid. As shown in Figures 24a (including insets), filamentous phages are the predominant morphology observed. This is complemented by the presence of tadpole-shaped phages, as depicted in Figure 24b. These findings collectively confirm that the phage population is polyclonal, with filamentous phages constituting the major type.

Transmission electron microscopy (TEM) in Figures 24c, 24d reveals the morphological diversity of bacteriophages infecting B. subtilis. The population includes both filamentous and tadpole-shaped phages, indicating a similarly mixed phage community as observed with E. coli.

Analysis: Quantitative analysis of the phage enrichment solutions (Spot titer assay) revealed that the E. coli phage lysate reached an endpoint dilution of 10^-6. The titer of the original solution was calculated as 5.6×10^8 PFU/mL based on the plaque count from the 10^-5 dilution. In parallel, the Bacillus subtilis phage lysate exhibited an endpoint dilution of 10^-7, with the plaque count at the 10^-6 dilution indicating an original concentration of 8.2×10^9 PFU/mL.

These findings indicate that the phage-enriched solutions contained not only highly lytic phages but also a diversity of temperate phages, which collectively contributed to effective and persistent suppression of bacterial growth.

We have successfully isolated environmental phages targeting E. coli and B. subtilis, and confirmed their lytic efficacy through planktonic bactericidal assays. These phages can serve as effective biological tools to precisely terminate the luteolin synthesis reaction.

Future work will focus on further purification of the phage isolates through continuous enrichment and screening, which is expected to enhance their lytic activity while eliminating potentially harmful contaminating phages, thereby improving overall safety. Furthermore, we plan to employ genetic engineering to augment phage functionality. Specifically, modification of phage genomes will not only enhance their bactericidal capacity for more reliable reaction termination but also, in combination with the Cre-loxP system, enable the delivery of cre recombinase genes. This integrated approach will establish a dual-control mechanism for regulating the synthetic pathway, effectively creating a programmable, phage-based switch.