Plasmid

Part:BBa_K5526007

Designed by: SIYUAN WU   Group: iGEM24_HWFLA-Beijing   (2024-09-02)


Plldr(New)-Azurin

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


BBa_K5526007 - Plldr(new)-Azurin

Composite Part: BBa_K5526007 (Plldr(new)-Azurin)

Construction Design

In the plasmid Plldr(new)-Azurin (BBa_K5526007), we combined Plldr-new (BBa_K5526001), Azurin (BBa_K5526000), and pUC57-mini (BBa_K3983004) to form Plldr(new)-Azurin (plactate2-Azurin). Plldr(new) has several improvements over the original promoter. It is more accurate and does not get inhibited by low oxygen concentrations. Azurin encodes a chemical drug that releases substances to kill tumor cells, while pUC57-mini serves as the plasmid backbone. Plldr(new)-Azurin is activated by high lactic acid concentrations and is unaffected by low oxygen levels, unlike the original.

Figure 1. The plasmid map of Plldr(new)-Azurin
Figure 1. The plasmid map of Plldr(new)-Azurin.

Engineering Principle

In this plasmid, we combined Plldr-new (BBa_K5526001), Azurin (BBa_K5526000), and pUC57-mini (BBa_K3983004) to create Plldr(new)-Azurin (plactate2-Azurin).

Experimental Approach

We performed PCR on the genes Azurin (444 bp) and the new pUC57-plldr (3800 bp). Agarose gel electrophoresis confirmed the lengths of these DNA fragments. The results showed that pUC57-plldr is 3800 bp, and Azurin is 444 bp.

Figure 2. Identification of PCR products by agarose gel electrophoresis
Figure 2. Identification of PCR products by agarose gel electrophoresis. Left: 3800 bp for pUC57-plldr. Right: 444 bp for p2-Azurin.

We used homologous recombination to combine Azurin with the new pUC57-plldr promoter, forming Plldr(new)-Azurin. We then performed a heat shock transformation on DH5α cells to facilitate plasmid uptake. After culturing the transformed cells on Amp+ medium, colonies grew, indicating successful plasmid uptake. Colony PCR confirmed the presence of the Plldr(new)-Azurin construct. Finally, the plasmids were sequenced to confirm the correct sequence.

Figure 3. PCR identification of plactate2-Azurin plasmid
Figure 3. PCR identification of plactate2-Azurin plasmid. A: 444 bp for p2-Azurin. B: Flora growing on petri dish. C: Sequencing results from the bio company.

Characterization/Measurement

We used alkaline lysis to extract plasmids from bacterial cultures and transformed them into EcN1917 competent cells using heat shock. Colony PCR verified the plasmid's transformation into EcN1917. Figure 4-A shows the results, with the amplified band at 550 bp indicating successful transformation.

Figure 4. PCR identification of EcN1917 transformants
Figure 4. PCR identification of EcN1917 transformants. A: Agarose gel showing 550 bp target band. B: Flora growing on a petri dish.

We grew bacteria containing the plasmids under different OD values (0.3, 0.6, 0.8, 1.0) and lactic acid concentrations (0 mM, 2 mM, 5 mM, 10 mM). Using a nanodrop, we measured the protein concentration and determined that the highest protein concentration occurred when OD600 was 0.6 and the lactic acid concentration was 5 mM. SDS-PAGE confirmed the desired protein expression.

Figure 5. Effects of bacterial concentration and lactic acid on plactate2-Azurin expression
Figure 5. Effects of bacterial concentration and lactic acid on plactate2-Azurin expression.

Other Tests

After identifying optimal expression conditions, we expanded the culture and used 5 mM lactic acid to induce the expression of the Azurin protein in EcN1917. SDS-PAGE showed a protein size of approximately 19 kDa, indicating successful expression of the Azurin protein (Figure 6).

Figure 6. Detection of Azurin protein expression by SDS-PAGE
Figure 6. Detection of Azurin protein expression by SDS-PAGE.

Summary

EcN drug molecular delivery carriers show promise due to their good compliance, long-lasting efficacy, and therapeutic precision. However, several challenges remain before they can be used in clinical settings. Future research will focus on animal experiments to assess the tumor inhibition effect of engineered probiotics in vivo. While current studies suggest that EcN strains may face challenges in clinical trials, further trials and studies are necessary to develop more effective and safer strains for use as probiotic drugs. Achieving precision tumor treatment using EcN drug molecular delivery vectors will require significant research and development.

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