Overview

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 microorganisms^1-3.

During our experiment, we added some new parts for iGEM part and new information to an existing part(Table 1), for instance, RIP (BBa_K4942011), pTRIP-GFP (BBa_K4942008) and pTRIP-ccdB (BBa_K4942010). Besides, we also made a contribution to some exiting parts, including pTrc99k (BBa_K3999002) which was redesigned to create a new composite part pTRIP (BBa_K4942006).

Table 1. Part contributions

1. Add New Experimental data to A Existing Part, BBa_K3999002 and Create a New Part BBa_K4942006

In order to construct the “switch” plasmid which could be transformed into E. coli to function, we used pTrc99k (BBa_K3999002) as the vector which was provided by SubCat.

We constructed pTRIP (pTrc99k-RIP) using homologous recombination.The pTrc99k plasmid was used as a template for PCR amplification, resulting in a fragment of 3408bp for pTrc. The Figure 1B shows a band consistent with the target size. The RIP sequence was amplified by PCR, with a length of 2065 bp. The Figure 1A indicates the band consistent with the results. It indicating successful amplification of RIP and linearization of the pTrc99k plasmid. After DpnI digestion and gel recovery, the pRIP fragment was obtained.

Figure 1. The gel electrophoresis validation of RIP and pTrc nucleic acids.

The plasmids were transformed into E.Coil DH5α . Figure 2A 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 2B and C, the pTRIP was successfully ligated to the pTrc99k vector without any apparent mutations, confirming the successful construction of the pTRIP plasmid.

Figure 2. The Monoclonal antibody validation and sequencing of pTRIP (pTrc99k-RIP, DH5α) .

Note：

A. Transformation plate of pTRIP:

B. Sequencing results of pTRIP

C. Base comparison of a specific region within pTRIP

2. Add a New Basic Part, BBa_K4942011 (RIP)

Length : 2062 bp

Source: synthetic

Usage and biology: the template sequences RIP include luxR2, luxI, PluxI, and PluxCDABEG. The consists of RIP is 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.

3. Add New Composite Parts, BBa_K4942008(pTRIP-GFP);BBa_K4942010(pTRIP-ccdB)

3.1 BBa_K4942008(pTRIP-GFP)

A.The construction of plasmid

We constructed the pTRIP-EGFP plasmid using homologous recombination. The EGFP sequence, which is 720bp in length, was amplified by PCR ( Figure 3A). Furthermore, using the pTRIP plasmid as a template, we performed PCR amplification and obtained 5473bp fragment of pTRIP-E (Figure 3 B).

Figure 3. The gel electrophoresis validation of EGFP and pTRIP-E .

We transformed the plasmid pTRIP-EGFP into DH5α, and Figure 4 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 showed at Figure 4 C. Subsequently, we sent clones 1-6 for sequencing. The sequencing results in Figure 4 D showed a 100% match with the nucleotide sequence of EGFP.

Figure 4. 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.

B. Protein expression

The target protein EGFP has a size of 26.9kDa. 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 5, 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 22 ^oC and deactivated state at 37 ^oC.

Figure 5.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 ^oC(DH5α)

C. Functional Test

Protein expression was induced by AI at a concentration of 0.6 mmol and at different temperatures. According to Figure 6, 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 6. 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 Figure7, 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 7.The fluorescence signal of report gene EGFP under microscope

Note:

A : pTRIP-EGFP 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 )

3.2 BBa_K4942010(pTRIP-ccdB)

A.The construction of plasmid

We constructed the pTRIP-ccdB plasmid using homologous recombination. The PCR amplification of the ccdB sequence resulted in a fragment of 306bp in length (figure 8).

Figure 8. The gel electrophoresis validation of ccdB.

By using the pTRIP plasmid as a template, we performed PCR amplification to obtain a 5473kb fragment (Figure 9) referred to as pTRIP-C.

Figure 9. 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 10A and B. Clones 1-8 were selected for antibody verification, and the results in Figure 10C demonstrate clear bands, confirming the presence of the ccdB sequence with a length of 306bp.

Next, colonies 1-8 were sent for sequencing, and the sequencing results in Figure 10 D showed a 100% match with the ccdB nucleotide sequence. It further validates the successful construction of the pTRIP-ccdB plasmid.

Figure 10. The monoclonal antibody validation and sequencing of pTRIP-ccdB (E.Coil DB3.1) .

B. 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 11 , 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 11 : The SDS-PAGE of ccdB protein

Note:

1-2: E.coil DB3.1(control)

3-4：37 ^oC-pTRIP-ccdB(E.coil DB3.1)

5-8：22 ^oC-pTRIP-ccdB(E.coil DB3.1)

9-10: E.coil DB3.1(control )

C . Functional Test

(1)The growth ability of pTRIP-ccdB (DH5α)

(1.1 )The pTRIP-ccdB transfer to DH5α

In order to verify the sensitivity of our temperature control system, we transferred the plasmid pTRIP-ccdB into DH5α. The length of ccdB is 306 bp, and Figure 12A proves that the colony growth is successful. The line 1-9 in Figure 17B are consistent with the expected results, which proves that pTRIP-ccdB has been successfully transformed into DH5α.

Figure 12. Verification of the pTRIP-ccdB (E.coil) monoclonal antibody

(1.2) The pTRIP-ccdB (E.coil DH5α) growth ability

a.The growth ability of pTRIP-ccdB (E.coil DH5α) at 22 ° C

According to Figure 13 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 13.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 Figure14A to E, we can summarize that the pTRIP-ccdB E.coil DH5α has a very similar growth trend with the control group ( 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 14.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 15, 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 15. Comparison of OD600 at 37°C and 22°C pTRIP-ccdB (E.coil DH5α ) with AI concentration of 0.6 at different times

(1.2) The growth ability of pTRIP-ccdB in E.coil BL21 (DE3).

(1.2.1)The pTRIP-ccdB transfor to E.coil BL21(DE3).

In order to verify the sensitivity of our temperature control system, we transferred the plasmid pTRIP-ccdB into E.coil BL21 (DE3). The length of ccdB is 306 bp, and Figure 16A proves that the colony growth is successful. The line 1-5 in Figure 16 B are consistent with the expected results, which proves that pTRIP-ccdB has been successfully transformed into E.coil BL21 (DE3).

Figure 16. Verification of the pTRIP-ccdB (E.coil BL21 (DE3)) monoclonal antibody

(1.2.2)The Growth ability 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 17, 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 17. The Growth ability of pTRIP-ccdB in E.coil BL21 ( DE3 )

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