Difference between revisions of "Part:BBa K5526005"
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Revision as of 14:37, 28 September 2024
Plldr-Azurin
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 335
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.
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.
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.
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.
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.
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.
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.