Plasmid

Part:BBa_K4942006

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


pTRIP


Documentation for BBa_K4942006

BBa_K4942006 (pTRIP, i.e. pTrc99k-RIP)

Construction Design

The BBa_K4942006 (pTRIP, i.e. pTrc99k-RIP) plasmid was constructed by synthetic BBa_K4942011 (RIP) from Genscript and the vector BBa_K3999002 (pTrc99k) provided by SubCat. The template sequences RIP include BBa_K4942001 (luxR2), BBa_K4942002 (luxI), BBa_K4942012 (PluxI), and BBa_K4942003 (PluxCDABEG). Homologous recombination method was employed for the construction of pTRIP (pTrc99k-RIP). The pTRIP functions as a temperature-controlled switch and consists of two fundamental regulatory proteins, LuxI and LuxR2.

Figure 1: The plasmid map of pTRIP

Figure 1. The plasmid map of pTRIP

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. Therefore, we constructed a temperature control system, pTRIP. 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 2: The engineering design schematic diagram

Figure 2. The engineering design schematic diagram.

Experimental Approach

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 backbone 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. The RIP sequence was amplified by PCR, with a length of 2065 bp. The Figure 3A indicates the band consistent with the results, indicating successful amplification of RIP and linearization of the pTrc99k plasmid. After DpnI digestion and gel recovery, the pRIP fragment was obtained.

Figure 3: The gel electrophoresis validation of RIP and pTrc nucleic acids

Figure 3. The gel electrophoresis validation of RIP and pTrc nucleic acids.

The plasmids were transformed into E. coli DH5α. Figure 4A 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 pTrc99k vector without any apparent mutations, confirming the successful construction of the pTRIP plasmid.

Figure 4: 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

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 5882
    Illegal BamHI site found at 288
    Illegal BamHI site found at 4258
    Illegal XhoI site found at 6226
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 484
    Illegal BsaI site found at 1552
    Illegal SapI.rc site found at 2634


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