Results

Figure 3. Diagram of CPTI circuit design

The TIA (trypsin inhibitor subtype A) gene was not successfully expressed (see Engineering Success Section). The proteinase inhibitor CPTI was chosen, and OmpA was selected as the signal peptide. OmpA has been successfully employed for extracellular expression in prokaryotic systems during laboratory work (Pechsrichuang et al., 2016, Movva et al., 1980). We also obtained information from the Peking University iGEM team, indicating their successful experience using OmpA as a signal peptide (see HP Section). Therefore, using OmpA as a signal peptide for CPTI expression appeared to be a more promising approach (Fig 3).

The OmpA -CPTI fragment (ordered from Guangzhou IGE Biotechnology Co.,Ltd.) was inserted into the pET-30a vector to generate the plasmid pET-30a-OmpA-CPTI. This plasmid was then transformed into E.coli BL21 and cultured overnight in LB medium. Overnight culture was dilute by 1/1000 to fresh LB medium, the IPTG was added when the O{D}_{600} reach 0.6. The medium was incubated another 3 hours after the ITPG addition, then centrifuged at 12,000 rpm 10 mins to obtain the supernatant and the precipitate. The supernatant was concentrated using ultrafiltration centrifuge tubes, while the precipitate was subjected to cell lysis with a lysis buffer. After lysis, the sample was centrifuged again to separate the supernatant from the post-lysis precipitate. The post-lysis precipitate was resuspended in lysis buffer. Tricine-PAGE (Fig 4) and Western Blot analyses (Fig 5) were performed on the above samples, and the results indicated that the CPTI protein was not expressed in E.coli.

Figure 4. Tricine-PAGE analysis of CPTI expression

Lane 1: Concentrated supernatant of OmpA-CPTI (+IPTG); Lane 2: Concentrated supernatant of OmpA-CPTI (-IPTG); Lane 3: Concentrated supernatant of pET-30a(+IPTG); Lane 4: Whole cell lysate of OmpA-CPTI (+IPTG); Lane 5: Whole cell lysate of OmpA-CPTI (-IPTG); Lane 6: Whole cell lysate of CPTI (+IPTG); Lane 7: Whole cell lysateof of CPTI (-IPTG); Lane 8: Whole cell lysate of pET-30a (+IPTG); Lane 9: Whole cell lysate of pET-30a (-IPTG)

Figure 5. Western blot analysis of CPTI expression

Lane 1: Concentrated supernatant of OmpA-CPTI (+IPTG); Lane 2: Concentrated supernatant of OmpA-CPTI (-IPTG); Lane 3: Concentrated supernatant of pET-30a(+IPTG); Lane 4: Whole cell lysate of OmpA-CPTI (+IPTG); Lane 5: Whole cell lysate of OmpA-CPTI (-IPTG); Lane 6: Whole cell lysate of CPTI (+IPTG); Lane 7: Whole cell lysate of of CPTI (-IPTG); Lane 8: Whole cell lysate of pET-30a (+IPTG); Lane 9: Whole cell lysate of pET-30a (-IPTG)

With IPTG the cell could produce the detectable CPTI (Lane 4, Fig 5), however, the concentrated supernatant of CPTI could not been seen (Lane 1, Fig 5). This might because of the poor solution of CPTI with OmpA singlal peptide. To get a extracellular expressed CPTI, the GST tag, which could improve the solution of certain protein, was successfully added at the C-terminal of the CPTI gene. The protein induction expression experiment is currently underway.

This modification ensures that the protein can be expressed to a significant extent, as similar experiments have been reported (Yang et al., 2003). Our unique approach involves testing the activity of CPTI without removing the GST tag, which distinguishes our study from previous ones.

Sequential Expression of Products for Delayed Toxin Release

The expression of Cry3A-Like toxin requires the activation of the first set of quorum sensing signal molecules. When engineered bacteria colonized in the gut, their population density is initially low, preventing the concentration of the first set of quorum sensing signal molecules from reaching the required threshold. Only CPTI is expressed and secreted to the gut environment (Fig 6).

As the engineered bacteria continue to be introduced and proliferate in the gut, the concentration of the first set of quorum sensing signal molecules gradually accumulates to the threshold level. This triggers the expression of Cry3A-like toxin and the onset of the second set of quorum sensing signal molecules. Upon reaching the threshold, the expression of the lysis gene is activated, leading to cell lysis, thereby releasing all Cry3A-like toxins into the environment.

The following gene circuit (first set of verification systems) is designed as follows

Figure 6. Diagram of first set of verification systems circuit design

To validate the sequential expression of products, the first set of verification systems were transformed to Top10 cells. Induction experiments were conducted using arabinose (Ara). The O{D}_{600} values, eGFP, and RFP fluorescence intensity were monitored at regular intervals using a microplate reader and plotted a curve with O{D}_{600} as the vertical axis and induction time as the horizontal axis. If the RFP signal appears with a delay after the eGFP signal, it indicates that the pathway can achieve sequential expression, confirming the success of the verification.

Implementation of Product Expression Control

Cry3A-like toxin and the second set of quorum sensing systems work with the same module, sharing the same promoter and the RBS of the same strength, and their expression levels are positively correlated. When the concentration of the second set of quorum sensing signal molecules has not reached the threshold level, both accumulate in the engineered bacteria. Once the threshold is reached, the lysis gene is actived, leading to cell lysis. In such a scenario, newly introduced engineered bacteria into the gut will promptly trigger the lysis gene, preventing the high-level expression of Cry3A-like toxin., which could significantly reduce the accumulation rate of Cry3A-like toxin in the environment, creating a situation similar to a "threshold" for its accumulation. A similar circumstance occurs for the accumulation of CPTI in the gut.

The following gene circuit (second set of verification systems) is designed as follows

Figure 7. Diagram of second set of verification systems circuit design

The second set of verification systems were transformed into Top10 cells and conducted induction experiments using Ara. At regular intervals, the O{D}_{600} values and eGFP fluorescence intensity were measured using a microplate reader. A curve with O{D}_{600} were plotted as the vertical axis and induction time were plotted as the horizontal axis. If the O{D}_{600} value were observed a significantly decreases after the initially increaseing, the expression of the lysis gene could be confrimed. We assessed whether the pathway could limit the maximum expression level based on the trend in eGFP signal changes. If the eGFP expression rate significantly decreases or ceases to increase after the appearance of lysis, the verification is successful. The limitation of the maximum expression level was assessed by the trend of eGFP signal change.

The complete sequences, synthesized (ordered from Guangzhou IGE Biotechnology Co.,Ltd.) and assembled onto respective plasmids, have been validated through sequencing. Both sets of plasmids were simultaneously introduced into E.coli Top10 using the KCM ice method. Successful transformation was confirmed by clone PCR.

Universal primers for the pBAD series plasmids were employed to confime the transformation of pBAD24M-paraBAD-esaⅠ-traR(W) or pBAD24M-paraBAD-lasⅠ-lasR. Amplification of bands corresponding to the size in pBAD24M for both sets of transformation plasmids resulted in bands of 1712 bp and 1845 bp (Fig 8), respectively. This confirms the successful introduction of the pBAD24M plasmids into both sets of plasmid systems.

Figure 8. Colony PCR of co-transformation by universal primers

Use pBAD24M forward primer and pBAD24M reverse primer as universal primers. Lane 1-2: Co-transformation of pBAD24M-paraBAD-esaⅠ-traR(W) and pBAD33-paraBAD-eGFP-ptra*-mCherry; Lane 3: Co-transformation of pBAD24M-paraBAD-lasⅠ-lasR and pBAD33-paraBAD-eGFP-plas-T4 holin-T4 endolysin

The PCR was also performed using specific primers to obtain target-sized fragments to confirms the successful introduction of pBAD33 plasmids into both sets of plasmid systems. The plasmid containing -paraBAD-eGFP-ptra*-mCherry yielded a 1224 bp fragment by using primers GCAGTGCTTCTCCCGTTAC/TTCCCAGCCCATAGTTTTC, while the plasmid containing paraBAD-eGFP-plas-T4 holin-T4 endolysin produced a 1296 bp fragment by using primers AGGCGGTCGTTGCTAATA/ AAACTCGTGCGGAGGTAA (Fig 9).

Figure. 9 Colony PCR of co-transformation by specific primers

Lane 1: pBAD33-paraBAD-eGFP-ptra*-mCherry; lane 2: Co-transformation of pBAD24M-paraBAD-esaⅠ-traR(W) and pBAD33-paraBAD-eGFP-ptra*-mCherry; lane 3: pBAD33-paraBAD-eGFP-plas-T4 holin-T4 endolysin; lane 4: Co-transformation of pBAD24M-paraBAD-lasⅠ-lasR and pBAD33-paraBAD-eGFP-plas-T4 holin-T4 endolysin

Validation of Product Expression Level Control Using the Second Verification System

Pre-experiment for Induced Expression of Lysis Effect

The successfully transformed engineered bacteria were streaked on plates and grown at 37°C. Single colonies were picked and inoculated into liquid LB medium, followed by the addition of inducers. The cultures were incubated for 6 hours.

Figure.10 Verification of lysis effect

The blank control was LB with 20% Ara. The control group consisted of the second verification system engineered bacteria without inducer, while the experimental group consisted of the second verification system engineered bacteria with a final concentration of 0.02% Ara. Each group had 3 replicates.

After zeroing with the blank control, it was observed that the optical density in the experimental group decreased to 34.3% of that in the control group (Fig 10), indicating a significant lysis effect. This confirms the successful expression of quorum sensing and initiation of downstream lysis gene expression. Lysis gene expression was successful without leakage.

Growth Curve Testing of the Second Verification System

Single clones were selected and inoculated into LB medium. After overnight incubation, the O{D}_{600} was adjusted to 0.6, and arabinose was added to a final concentration of 0.02%. The cultures were shaken for 21 hours in a sterile 96-well plate, and a growth curve was plotted.

The blank control group was LB broth, the control group was wild Top10, and the experimental group was the second set of engineering bacteria for verification system. There were 6 replicates per group.

Figure 11. Growth curve testing of the second set of verification system

The growth curve revealed that the bacterial density continued to rise in the first 4 hours and began to decrease after 4 hours, stabilizing around the 9th hour. In contrast, the control group's bacterial density continued to rise. At the 4th hour, the quorum sensing signal reached the threshold, initiating lysis gene expression. The engineered bacteria lysed, resulting in a significant decrease in bacterial density, which stabilized around the 9th hour (Fig 11).

Characterization of Reporter Gene eGFP

Single colonies of engineered bacteria were selected and inoculated into LB broth. After overnight growth, the O{D}_{600} was adjusted to 0.6. Ara at a final concentration of 0.02% was added, and the cultures were incubated for 6 hours. eGFP fluorescence intensity was measured using a microplate reader (excitation wavelength 488 nm, emission wavelength 506 nm).

Blank control group: sterile LB, control group: engineered bacteria without 20% ara added, experimental group: engineered bacteria with inducer added, 3 replicates per group.

There was no significant difference in fluorescence intensity between the control group and the experimental group, indicating no expression of eGFP (data not shown).

eGFP Protein Expression Experiment

Single colonies of engineered bacteria were selected and inoculated into LB medium with inducer for overnight incubation. Top10 subjected to the same procedure served as the control group. Bacterial pellets were collected, and SDS-PAGE experiments were conducted, revealing no expression of eGFP at the protein level(data not shown).

q-RT-PCR Experiment

q-RT-PCR experiments were conducted under various conditions, confirming no expression at the mRNA level.

The reporter genes could not be expressed. The characterization of reporter genes eGFP and RFP was performed simultaneously with the second verification system. Consistent results were obtained from SDS-PAGE experiments and q-RT-PCR experiments (data not shown).

We hypothesize several possible reasons:
1. The selected reporter genes eGFP and RFP may not be suitable for expression in Top10.
2. Errors may have occurred in the design of the gene expression module.
3. Interactions between genes may have occurred.

Extraction and Detection of Quorum Sensing Signal Molecules:

According to the literature, High-performance liquid chromatography (HPLC) can be used to detect C_4-HSL(Ma et al., 2017). HPLC were utilized to determine the retention times and signals of 3-oxo- C_6-HSL and 3-oxo-C_{12}-HSL standards, demonstrating the applicability of HPLC for detecting both substances (Fig12-13). Standard curves correlating concentration and peak area were constructed for both 3-oxo-C_6-HSL and 3-oxo- C_{12}-HSL (Fig 17).

Construction of the 3-oxo-C_{12}-HSL Standard Curve:

Standard samples with concentrations of 0.3125 nm, 0.625 nm, 1.25 nm, 2.5 nm, and 5 nm were used for three replicates at each concentration. The mean values were then used to generate the standard curve.

Figure 12. Chromatographic diagram of molecule 3-oxo-C_6-HSL

Figure 13. Chromatographic diagram of molecule 3-oxo-C_{12}-HSL

Figure 14. Standard curve for molecule 3-oxo-C_{12}-HSL

y=4173346.303x+341501.737 R^2=0.997

Extraction and Detection of E.coli Signal Molecules

Inoculate seed culture with an O{D}_{600} of 0.6 into 24 conical flasks containing 100 mL LB each. 1% ara was added to induce expression and cultures were shaked at 37°C and 220 rpm. At 1-hour intervals, samples were collected from 3 flasks, labeled as Sample 1, Sample 2, and Sample 3. 20 mL of bacterial culture were extracted from each flask and centrifuged at 4000 rpm for 10 minutes. The supernatant were treated with an equal volume of ethyl acetate for 4 hours. The supernatant was centrifuged and evaporated to dry. The samples were stored at -20°C for subsequent HPLC analysis.

Figure 15. Chromatographic diagram of molecule 3-oxo-C_6-HSL from Pseudomonas syringae

The quorum sensing signal molecule 3-oxo-C_6-HSL from Pseudomonas syringae was detected using this method (Fig 15).

However, when the same approach was applied to the engineered bacteria, we encountered issues with multiple impurities in the chromatograms and the inability to detect the signal (Fig 20-21). Since the second validation system successfully lysed without leakage, it is certain that the quorum sensing signal molecule 3-oxo-C_{12}-HSL was produced and reached the threshold in the extracellular environment. After two rounds of induction and extraction experiments, this method for detecting signal molecule production were abandoned.

Figure 16. Result of the first set of validation system induced for 8 hours

Figure 17. Result of the second set of validation system induced for 8 hours

Validation of Sequential Expression of Products Using the First Set of Validation System

The characterization of reporter genes eGFP and RFP was performed simultaneously with the second validation system. The results were consistent, showing no expression in SDS-PAGE and q-RT-PCR experiments.

We will continue to attempt changing the expression vector or reporter genes to complete the functional validation of the secretion gene pathway. We will replace eGFP and RFP with the validated protease inhibitor and toxin proteins, assemble the complete gene pathway, and perform testing. Finally, the complete gene pathway will be introduce into the chromosome of Top10 for further evaluation.