Plasmid

Part:BBa_K4942010

Designed by: Huang Xinqi   Group: iGEM23_SHSID-China   (2023-08-10)


pTRIP-ccdB


BBa_K4942010 Documentation

BBa_K4942010

Introduction

BBa P1011 ccdB encodes a protein toxic to most strains of Escherichia coli. In our team, by combining the ccdB protein expression and the temperature-based regulation engineering of pTRIP (BBa_K4942006), we can form a temperature-based “kill-switch,” pTRIP-ccdB (BBa_K4942010), thereby preventing the leakage of engineered microorganisms, which expands the usage of ccdB and makes a contribution to the biosafety career.

Construction Design and Engineering Principle

Some genetically modified microorganisms used in the production of engineered probiotics or industrial fermentation strains require special precautions for biosafety[1]. 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 contamination[2]. 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[3]. We constructed a temperature control system (pTRIP), and 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

Figure 1. The engineering design schematic diagram.

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 redesign ccdB by introducing it into pTRIP to upgrade the toxic protein to a “kill-switch” (Figure 2). The recombinant plasmid pTRIP-ccdB was obtained by homologous recombination.

Figure 2: The plasmid map of pTRIP-ccdB

Figure 2. The plasmid map of pTRIP-ccdB

Experimental Approach

1. Construction of pTRIP-ccdB 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 3 indicates that the amplified band matches the expected size, confirming the successful amplification of the ccdB sequence from the linearized plasmid.

Figure 3: The gel electrophoresis validation of ccdB.

Figure 3. 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 4 shows that the amplified band matches the expected size, indicating the successful amplification of the linearized pTRIP-C plasmid.

Figure 4: The gel electrophoresis validation of pTRIP-C.

Figure 4. 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 5 A and B. Clones 1-8 were selected for antibody verification, and the results in Figure 5C demonstrate clear bands, confirming the presence of the ccdB sequence with a length of 306bp. The gel electrophoresis image in Figure 4C matches the expected band, indicating the successful transformation.

Figure 5: The monoclonal antibody validation and sequencing of pTRIP-ccdB (E.Coil DB3.1).

Figure 5. The monoclonal antibody validation and sequencing of pTRIP-ccdB (E.Coil DB3.1).

Next, colonies 1-8 were sent for sequencing, and the sequencing results in Figure 5D 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.

2. 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 Figure 6, 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 6: The SDS-PAGE of ccdB protein

Figure 6. The SDS-PAGE of ccdB protein

Note:
1-2: E. coli DB3.1 (control)
3-4: 37oC-pTRIP-ccdB (E. coli DB3.1)
5-8: 22oC-pTRIP-ccdB (E. coli DB3.1)
9-10: E. coli DB3.1 (control)

Characterization/Measurement

On the basis of BBa _ P1011 ( ccdB ), we constructed a new combination plasmid BBa _ K4942010 ( pTRIP-ccdB ), and transformed the plasmid into E. coli DH5α and E. coli BL21 (DE3). The growth ability of pTRIP-ccdB at different temperatures of 22 °C and 37 °C by AHL induction was verified, and the sensitivity of the temperature control switch was further verified.

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

A. The growth ability of pTRIP-ccdB (E. coli DH5α) at 22 °C
According to Figure 7 A to E, given the temperature 22 °C, the OD600 of E. coli 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 in 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 7: The growth ability of pTRIP-ccdB (E. coli DH5α) in different AI concentrations at 22 °C

Figure 7. The growth ability of pTRIP-ccdB (E. coli DH5α) in different AI concentrations at 22 °C

B. The growth ability of pTRIP-ccdB (E. coli DH5α) at 37 °C
According to Figure 8A-E, the pTRIP-ccdB E. coli DH5α has a very similar growth trend to the control group, the wild E. coli 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 8: The growth ability of pTRIP-ccdB (E. coli DH5α) in different AI concentrations at 27 °C

Figure 8. The growth ability of pTRIP-ccdB (E. coli DH5α) in different AI concentrations at 27 °C

C. Comparison of the growth ability of pTRIP-ccdB (E. coli DH5α) at 37 °C and 22 °C
According to Figure 9, 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. coli 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 9: Comparison of OD600 at 37°C and 22°C pTRIP-ccdB (E. coli DH5α) with AI concentration of 0.6 at different times

Figure 9. Comparison of OD600 at 37°C and 22°C pTRIP-ccdB (E. coli DH5α) with AI concentration of 0.6 at different times

2. The growth ability test of pTRIP-ccdB in E. coli BL21 (DE3)

Comparisons of growth capabilities were made at 37°C and 22°C with an AI concentration of 0.6 mmol. The E. coli BL21 was the control group. According to Figure 10, 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 a similar value. This test result is consistent with that for DH5α as discussed previously and this also backs up the engineering success of our temperature-based “kill switch,” the plasmid pTRIP-ccdB in E. coli host.

Figure 10: The Growth ability of pTRIP-ccdB in E. coli BL21 (DE3)

Figure 10. The Growth ability of pTRIP-ccdB in E. coli BL21 (DE3)

Reference

  1. Bazhenov, S.V., Scheglova, E.S., Utkina, A.A. et al. New temperature-switchable acyl homoserine lactone-regulated expression vector. Appl Microbiol Biotechnol 107, 807–818 (2023). https://doi.org/10.1007/s00253-022-12341-y
  2. Nocadello, S., Swennen, E.F. The new pLAI (lux regulon based auto-inducible) expression system for recombinant protein production in Escherichia coli. Microb Cell Fact 11, 3 (2012). https://doi.org/10.1186/1475-2859-11-3
  3. Hoffmann SA, Diggans J, Densmore D, Dai J, Knight T, Leproust E, Boeke JD, Wheeler N, Cai Y. Safety by design: Biosafety and biosecurity in the age of synthetic genomics. iScience. 2023 Feb 10;26(3):106165. https://doi.org/10.1016/j.isci.2023.106165


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 5103
    Illegal BamHI site found at 3479
    Illegal XhoI site found at 5447
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1866


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