Plasmid

Part:BBa_K4942008

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


pTRIP-EGFP


Documentation for BBa_K4942008

BBa_K4942008

Construction Design

We constructed a temperature control system (pTRIP) and introduced a visual reporting gene EGFP (BBa_K3521004) into the system to monitor its functionality and optimize its sensitivity.

The 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.

Figure 1: The plasmid map of pTRIP-EGFP

Figure 1. The plasmid map of pTRIP-EGFP

Engineering Principle

Some genetically modified microorganisms used in the production of engineered probiotics or industrial fermentation strains require special precautions for biosafety. 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. 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.

Figure 2: The engineering design schematic diagram

Figure 2. The engineering design schematic diagram.

Cultivation, Protein Purification and SDS-PAGE

1. The construction of pTRIP-EGFP plasmid

We constructed the pTRIP-EGFP plasmid using homologous recombination. The EGFP sequence, which is 720bp in length, was amplified by PCR. The Figure 3A 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 a 5473bp fragment of pTRIP-E. The Figure 3B 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 3: The gel electrophoresis validation of EGFP and pTRIP-E

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

We transformed the plasmid pTRIP-EGFP into DH5α, and Figure 4A 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 4C showed bands that matched the expected size, indicating successful transformation. Subsequently, we sent clones 1-6 for sequencing. The sequencing results in Figure 4D 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 4: The Monoclonal antibody validation and sequencing of pTRIP-EGFP (E.Coil DH5α)

Note:

A. Transformation plate of pTRIP-EGFP:

B. Sequencing results of pTRIP-EGFP

C. Base comparison of a specific region within pTRIP-EGFP

2. 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 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 expressed 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 5: The SDS-PAGE protein gel of EGFP at different temperatures

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)

Characterization/Measurement

Protein expression was induced by AI at a concentration of 0.6 mmol and at different temperatures. As shown in 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 stage.

Figure 6: The fluorescence intensity of reporter gene EGFP at different temperatures

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 Figure 7, 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 an obvious fluorescence signal. It shows that there is no fluorescence signal at 37 oC, and an obvious fluorescence signal can be seen at 22 oC.

Figure 7: The fluorescence signal of report gene EGFP under microscope

Figure 7. The fluorescence signal of report gene EGFP under the microscope

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 5823
    Illegal BamHI site found at 4199
    Illegal XhoI site found at 6167
  • 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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