Some genetically modified microorganisms used in the production of engineered probiotics or industrial fermentation strains require special precautions for biosafety1. It is important to prevent the unintentional release, multiplication, and spread of these genetically modified microorganisms into the environment, which could lead to unpredictable biological contamination2. This project has designed a simple and user-friendly "safety lock" for engineered microorganisms. Under normal conditions at 37℃ (the temperature inside the human body, which is also the working temperature for probiotics and commonly used in industrial microbial fermentation), the "safety lock" remains inactive, allowing the host microorganism to reproduce and function normally. However, at 22℃ (a temperature closer to natural environmental conditions, excluding tropical regions and extremely hot summers), the "safety lock" becomes active, expressing a toxic protein that leads to the self-destruction of the host microorganism, thereby preventing the release of the engineered microorganisms3-5. We constructed a temperature control system（pTRIP）, and introduced a visual reporting gene(EGFP), into the system to monitor its functionality and optimize its sensitivity. Lastly, we developed a plasmid, pTRIP-ccdB, which contains a toxic protein, (ccdB), that can kill bacteria upon expression, thereby preventing the leakage of engineered microorganisms(Figure 1).
.
Figure 1. The engineering design schematic diagram.
Design:
pTRIP plasmid was constructed by synthetic RIP from Genscript and the vector ppTrc99k.provided by SubCat.
The template sequences RIP include luxR2, luxI, PluxI, and PluxCDABEG. Homologous recombination method was employed for the construction of pTRIP (ppTrc99k-RIP).
The pTRIP functions as a temperature-controlled switch and consists of two fundamental regulatory proteins, LuxI and LuxR2. LuxI is a synthesis enzyme for the autoinducer, which generates signal molecules known as AHL or HSL. LuxR, on the other hand, serves as both a receiver for cytoplasmic signal molecules and a DNA-binding transcriptional regulator. These two proteins jointly regulate the expression of the bioluminescence-related fluorescent protein manipulator, PluxCDBEG. Consequently, it allows for the control of both the PluxCDBEG module and protein expression.
In order to measure the function effectivity of our system, we induced the reporter EGFP for visible fluoresces which was also provided by SubCat. Using pTRIP plasmid as a template, the EGFP fragment was homologously recombined with a linearized plasmid to construct the pTRIP-EGFP plasmid.
Figure 2. The plasmid map of pTRIP-EGFP
Build:
1.1 pTRIP
We constructed pTRIP using homologous recombination. The RIP sequence was amplified by PCR, with a length of 2065 bp. The Figure 3A indicates the band consistent with the results. The pTrc99k plasmid was used as a template for PCR amplification, resulting in a fragment of 3408bp for pTrc. The Figure 3B shows a band consistent with the target size. It indicating successful amplification of RIP and linearization of the pTrc plasmid. After DpnI digestion and gel recovery, the pRIP fragment was obtained.
Figure 3. The gel electrophoresis validation of RIP and pTrc nucleic acids.
The plasmids were transformed into E.Coil DH5α . Figure4 A shows the presence of single colonies on the plate. We selected colonies 1-10 and sent them directly for sequencing. According to the results shown in Figure 4B and C, the pTRIP was successfully ligated to the pTrc vector without any apparent mutations, confirming the successful construction of the pTRIP plasmid.
Figure 4. The Monoclonal antibody validation and sequencing of pTRIP (DH5α) .
Note：
A. Transformation plate of pTRIP:
B. Sequencing results of pTRIP
C. Base comparison of a specific region within pTRIP
1.2 pTRIP-EGFP
We constructed the pTRIP-EGFP plasmid using homologous recombination. The EGFP sequence, which is 720bp in length, was amplified by PCR. The Figure 5 A showed that matched the expected size, indicating successful amplification of the EGFP sequence. Furthermore, using the pTRIP plasmid as a template, we performed PCR amplification and obtained 5473bp fragment of pTRIP-E. The Figure 5 B showed bands that matched the expected size, indicating successful amplification of the linearized pTRIP-E plasmid. We can conclude that we successfully amplified the EGFP sequence and linearized pTRIP-E plasmid.
Figure 5. The gel electrophoresis validation of EGFP and pTRIP-E .
We transformed the plasmid pTRIP-EGFP into DH5α, and Figure 6 A and B showed the growth of single clones. We selected clones 1-6 and performed antibody verification.The expected size of 720bp for the EGFP sequence. Additionally, Figure 6 C showed bands that matched the expected size, indicating successful transformation. Subsequently, we sent clones 1-6 for sequencing. The sequencing results in Figure 6 D showed a 100% match with the nucleotide sequence of EGFP, confirming the successful integration of the EGFP fragment into the pTRIP-E plasmid and further validating the successful construction of the plasmid.
Figure 6. The Monoclonal antibody validation and sequencing of pTRIP-EGFP (E.Coil DH5α) .
Note:
A.B shows the plate images of pTRIP-EGFP transformed into E.Coil DH5α cells.
C is the image showing the verification of single clone colonies.
D is the sequencing image of pTRIP-EGFP plasmid.
Test:
1.Protein expression
The target protein EGFP has a size of 26.9kDa. Protein expression was induced at a concentration of 0.6mmol AI (AutoInducers, herein it refers to N-(3-oxohexanoyl)-L-homoserine lactone), and induction was performed at 37 oC and 22 oC. According to the figure 9, in the control group (line 1), no EGFP protein is present. Under the condition of 37 oC (line 1 to line 3), EGFP protein was not observed. However, under the condition of 22 oC (line 4 to line 5), there is a clear presence of EGFP protein. This indicates that EGFP expression is not express or occurring at a lower level at 37 oC, while there is substantial expression at 22 oC. Therefore, it can be concluded that our temperature control system is activated state at 22oC and deactivated state at 37 oC.
Figure 7.The SDS-PAGE protein gel of EGFP at different temperatures
Note:
line 1: pTRIP(DH5α)
line 2：pTRIP-EGFP-37 oC(DH5α)
line 3：pTRIP-EGFP-37 oC(DH5α)
line 4：pTRIP-EGFP-22 oC(DH5α)
line 5：pTRIP-EGFP-22 oCo(DH5α)
2. Functional Test
3.1.1 Detection of fluorescence intensity EGFP
Protein expression was induced by AI at a concentration of 0.6 mmol and at different temperatures. As shown in Figure 8, the fluorescence intensity of pTRIP-EGFP increased first and then decreased with the increase of temperature. At 22 oC, the fluorescence intensity of pTRIP-EGFP was the strongest. At 37 oC, the fluorescence intensity of pTRIP-EGFP was the weakest. However, no fluorescence signal was detected in the control group pTRIP. This shows that the temperature control system of our subject is controlled by temperature. At 22 oC, in the open state ; At 37 oC, it is close to the closed state.
Figure 8. The fluorescence intensity of reporter gene EGFP at different temperatures
In order to observe the fluorescence signal of pTRIP-EGFP more clearly, we made the glass slides of pTRIP-EGFP bacteria and control group ( pTRIP ) cultured at 22 oC and 37 oC. According to the Figure9, A (colonies under white light) and B (pTRIP-EGFP at 37 oC) have no fluorescence signal, C and D (pTRIP-EGFP at 22 oC) have obvious fluorescence signal. It shows that there is no fluorescence signal at 37 oC, and obvious fluorescence signal can be seen at 22 oC.
Figure 9.The fluorescence signal of report gene EGFP under microscope
Note:
A : Colonies under white light
B. Fluorescence reporter gene of pTRIP-EGFP plasmid at 37 °C
C. 22 ° fluorescence reporter gene of pTRIP-EGFP plasmid ( 10 × 40 )
D. 22 ° fluorescence reporter gene of pTRIP-EGFP plasmid ( 10 × 100 )
Learn:
Through this cycle that evaluated the function of the pTRIP-EGFP system, we can basically predict that the temperature-based “switch” works so that we can step further to construct the temperature-based “kill-switch” system.
Besides, we also encourage future iGEM teams to further explore and optimize pTRIP-EGFP, such as whether it is a self-induced temperature control system or a system that requires exogenous induction, which requires more experimental data for more precise evaluation, for example, the relationship between EGFP fluorescence intensity and temperature can be more refined, the temperature gradient can be more intensive, and the optimal induction time also needs to be further explored.
Design:
The ccdB is a toxic protein that needs to be transformed into E.coli DB3.1 competent cells. E.coli DB3.1 competent cells are anti-toxic. In this cycle, we will replace EGFP with ccdB to upgrade the temperature-based “switch” to a “kill-switch”(Figure 10).
After the first cycle, we successfully obtained pTRIP plasmid which functions as a temperature-controlled switch.The pTRIP-C linearized plasmid was obtained by using pTRIP plasmid as template.
Figure 10 . The plasmid map of pTRIP-ccdB
Build:
2.1 pTRIP-ccdB
We constructed the pTRIP-ccdB plasmid using homologous recombination. The PCR amplification of the ccdB sequence resulted in a fragment of 306bp in length. The figure 11 indicates that the amplified band matches the expected size, confirming the successful amplification of the ccdB sequence from the linearized plasmid.
Figure 11. The gel electrophoresis validation of ccdB.
By using the pTRIP plasmid as a template, we performed PCR amplification to obtain a 5473kb fragment referred to as pTRIP-C. The gel electrophoresis image in Figure 12 shows that the amplified band matches the expected size, indicating the successful amplification of the linearized pTRIP-C plasmid.
Figure 12. The gel electrophoresis validation of pTRIP-C.
The pTRIP-ccdB plasmid was transformed into E. coli DB3.1. The single clone colony growth on plates is shown in Figure 13 A and B. Clones 1-8 were selected for antibody verification, and the results in Figure 15C demonstrate clear bands, confirming the presence of the ccdB sequence with a length of 306bp. The gel electrophoresis image in Figure 15C matches the expected band, indicating the successful transformation.
Next, colonies 1-8 were sent for sequencing, and the sequencing results in Figure 13 D showed a 100% match with the ccdB nucleotide sequence. This confirms the successful integration of the ccdB fragment into the pTRIP-E plasmid. It further validates the successful construction of the pTRIP-ccdB plasmid.
Figure 13. The monoclonal antibody validation and sequencing of pTRIP-ccdB (E.Coil DB3.1) .
Test:
1.Protein expression
The size of the ccdB protein, the target protein, is 11.7 kDa. Protein expression was induced at a concentration of 0.6mmol AI, and induction was performed at 37 oC and 22 oC. According to the figure 14, in the control group (line 1 -line 2 and line 9 -line 10), ccdB protein was not observed. Under the condition of 37 oC (line 3 to line 4), ccdB protein was not observed. However, under the condition of 22 oC (line 5 to line 8), a weak presence of ccdB protein is detected. This indicates that ccdB expression does not occur at 37 oC, while there is limited expression at 22 oC. Therefore, it can be concluded that our temperature control system is in the activated state at 22 oC and in the deactivated state at 37 oC.
Figure 14 : The SDS-PAGE of ccdB protein
Note:
1-2: E.coil DB3.1(control)
3-4：37oC-pTRIP-ccdB(E.coil DB3.1)
5-8：22oC-pTRIP-ccdB(E.coil DB3.1)
9-10: E.coil DB3.1(control )
2. The growth ability test of pTRIP-ccdB (DH5α)
A. The growth ability of pTRIP-ccdB (E.coil DH5α) at 22 ° C
According to Figure 15 A to E, given the temperature 22 °C, the OD600 of E.coil in the control group increased first and then gradually tended to be stabilized over time. While there was no significant increase in OD600 of pTRIP-ccdB at the experimental group over time. Therefore, compared with the wild DH5α (the control group), we can conclude that the ccdB did kill bacteria, the host at 22 °C upon expression.
Figure 15.The growth ability of pTRIP-ccdB ( E.coil DH5α ) in different AI concentrations at 22 ° C
B. The growth ability of pTRIP-ccdB (E.coil DH5α) at 37 ° C
According to Figure16A-E, the pTRIP-ccdB E.coil DH5α has a very similar growth trend with the control group, the wild E.coil DH5α at 37 °C which means the “switch” pTRIP-ccdB has little effect on the strain’s growth since no expression of ccdB at 37 °C.
Figure 16.The growth ability of pTRIP-ccdB ( E.coil DH5α ) in different AI concentrations at 27 ° C
C. Comparison of the growth ability of pTRIP-ccdB (E.coil DH5α) at 37 °C and 22 °C
According to Figure 17, in two groups with AI concentration of 0.6 mmol, the OD600 of pTRIP-ccdB at 37 °C increased firstly and then tended to be stabilized over time, while there was almost no significant change of that at 22 °C. It is seen that pTRIP-ccdB (E.coil DH5α) grew much better at 37 °C than 22 °C where it indicated little growth over time. This further supports the conclusion that the bacterial strain grows normally at 37°C, while the presence of ccdB at 22°C leads to bacterial cell death.
.
Figure 17. Comparison of OD600 at 37°C and 22°C pTRIP-ccdB (E.coil DH5α ) with AI concentration of 0.6 at different times
2.2 The growth ability test of pTRIP-ccdB in E.coil BL21 (DE3).
Comparisons of growth capabilities were made at 37°C and 22°C with an AI concentration of 0.6 mmol.The E.coil BL21 was the control group. According to Figure 18, it is evident that the OD600 of BL21 (the control group) is significantly higher than that of BL21(pTRIP-ccdB) at 22°C while the OD600 of BL21 and BL21(pTRIP-ccdB) have similar value. This test result is consistent with that for DH5α as discussed previously and this also back up the engineering success of our temperature-based “kill switch”, the plasmid pTRIP-ccdB in E. coli host.
Figure 18. The Growth ability of pTRIP-ccdB in E.coil BL21 ( DE3 )
Learn:
1. Based on the growth ability tests, it indicates that the experimental strains with pTRIP-ccdB could grow normally at 37 °C but stop growing at 22 °C which validates the engineering success of our temperature-based “kill-switch” as expected.
2. In the experiment, we only conducted sensitivity testing of the temperature-controlled switch in E. coli BL21(DE3) and E. coli DH5α. In the future, we need to transform it into more bacterial strains to perform sensitivity tests and further optimize the sensitivity of our temperature-controlled switch based on the test results. We also need to explore the temperature range of 22-37°C for the temperature-controlled switch and validate its states through additional verification methods.
3. Our temperature-controlled switch requires the addition of exogenous inducers (N-(3-oxohexanoyl) -L-homoserine lactone) for better expression. When no inducer is added, the expression level is relatively low, and this limitation needs to be addressed in practical applications.