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Revision as of 06:07, 29 September 2024

Plldr-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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 335


Plldr-Azurin (BBa_K5526005) Documentation

Composite Part: BBa_K5526005 (Plldr-Azurin)

Construction Design

In the plasmid Plldr-Azurin (BBa_K5526005) we constructed, we combined Plldr (BBa_K822000), Azurin (BBa_K5526000), and pUC57-mini (BBa_K3983004) to form Plldr-Azurin (plactate 1-Azurin). Plldr is a lactic acid promoter that will be activated under a high lactic acid concentration. Azurin is a gene that encodes a chemical drug, which can release some medicines and substances to kill the tumor cells. pUC57-mini is the skeleton of the plasmid. Plldr-Azurin is the plasmid that will be activated and produce a toxic protein with a high lactic acid concentration.

Plasmid map of Plldr-Azurin
Figure 1. The plasmid map of Plldr-Azurin.

Engineering Principle

In the plasmid Plldr-Azurin (BBa_K5526005) we constructed, we combined Plldr (BBa_K822000), Azurin (BBa_K5526000), and pUC57-mini (BBa_K3983004) to form Plldr-Azurin (plactate 1-Azurin).

Experimental Approach

We applied PCR on the genes Azurin (444bp) and pUC57-plldr (3150bp); we used agarose gel electrophoresis to check the length of our PCR production to ensure we succeeded. The result shows that the pUC57-plldr (plactate 1) has a length of 3150bp, and the Azurin has a length of 444bp.

PCR production identification by agarose gel electrophoresis
Figure 2. The identification of PCR production by agarose gel electrophoresis. Left: The graph shows that plactate 1 has a length of 3150 bp. Right: The graph shows p1-Azurin has a length of 444bp.

We first used homologous recombination to combine Azurin with the lldr promoter, forming the Plldr-Azurin (plactate1-Azurin). We then performed a heat shock conversion to make DH5α cells sensitive to frequent changes in temperature, alternating between high and low temperatures to facilitate the uptake of plasmids of DH5α. After heat shock, we injected the plasmids into DH5α cells and grew them on an Amp+ medium, ensuring that only bacteria containing the plasmids would survive. As expected, bacterial colonies grew on the petri dishes, indicating successful plasmid uptake. To further confirm the presence of the desired plasmid, we performed a colony PCR directly from the colonies on the plate. This allowed us to amplify the specific region of the plasmid containing the Plldr-Azurin (plactate1-Azurin) construct. Figure 3 shows the PCR results were positive, indicating that the colonies contained the correct plasmid. Finally, we recycled the plasmids and sent them for sequencing at a bio company to ensure the correct sequence. The sequencing results confirmed that the plasmids were indeed the ones we wanted, with the correct sequence and no mutations.

PCR identification of plactate1-Azurin plasmid
Figure 3. PCR identification of plactate1-Azurin plasmid. A. The graph shows the length of p1-Azurin is 444bp. B. The graph shows the flora growing in a petri dish. C. The graph shows the sequencing measured by the bio company.

Characterization/Measurement

We used alkaline lysis to extract plasmids (plactate1-Azurin) from bacterial cultures. Next, we converted the recombinant plasmid to EcN1917 competent by heat shock transformation. PCR was used to verify whether the plasmid was transformed into EcN1917. The results are shown in Figure 4-A. The colony PCR results were detected by agarose gel electrophoresis, and the amplified band was 550bp, which was consistent with the target band, indicating that the four plasmids were successfully transformed into EcN1917.

PCR identification of EcN1917 transformants
Figure 4. PCR identification of EcN1917 transformants. A. The PCR production of the plactate1-Azurin transformants by agarose gel electrophoresis. B. The graph shows the flora growing in a petri dish.

We grew the bacteria holding the plasmids under different OD values (0.3, 0.6, 0.8, 1.0) and lactic acid concentrations (0mM, 2mM, 5mM, 10mM). We used the nanodrop to detect protein concentration and created a graph of four different bacteria’s protein expression. We concluded that the protein concentration is highest when OD600 equals 0.6 and the lactic acid concentration is 5mM. We used SDS-PAGE to ensure we got the proteins we wanted.

Effects of bacterial concentration and lactic acid concentration on protein expression
Figure 5. The effects of bacterial concentration and lactic acid concentration on the expression of plactate1-Azurin.

Other Tests

After the optimal protein expression conditions were obtained, the constructed strains were expanded for culture, and 5 mM lactic acid was used to induce the actual expression level of tumor drugs in EcN1917. The experimental results are shown in Figure 6. The size of the Azurin protein is about 19kDa. A large amount of target protein indicates that the Azurin protein is successfully expressed. In the future, we will further study the expression and tumor inhibition effect of the constructed probiotics in the tumor environment.

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

Summary

EcN drug molecular delivery carriers are characterised by good compliance, long-lasting efficacy, and therapeutic precision. However, many issues still need to be resolved before they can be placed on the market. We will conduct animal experiments in the future to verify the tumor inhibition effect of our engineered probiotics in animals. Existing studies have shown that EcN strains may not be ideal in clinical trials, so we need more trials and studies to produce more effective and safer EcN strains that can be used as probiotic drugs. Therefore, there is still a long way to go to achieve precision treatment of tumors using EcN drug molecular delivery vectors.