Difference between revisions of "Part:BBa K5526007"
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     <title>Plldr(new)-antiPD-L1 (BBa_K5526007) Documentation</title>
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     <title>BBa_K5526007 - Plldr(new)-Azurin</title>
 
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     <h2>Composite Part: BBa_K5526007 (Plldr(new)-Azurin)</h2>
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     <h2>Composite Part: BBa_K5526007 (Plldr(new)-antiPD-L1)</h2>
+
  
 
     <h3>Construction Design</h3>
 
     <h3>Construction Design</h3>
−
     <p>In the plasmid Plldr(new)-antiPD-L1 (BBa_K5526007) we constructed, we combined Plldr-new (BBa_K5526001), antiPD-L1 (BBa_K5526008), and pUC57-mini (BBa_K3983004) to form Plldr(new)-antiPD-L1 (plactate2-antiPD-L1). Plldr-new includes several improvements to the original promoter, making it more accurate and resistant to inhibition under low oxygen concentrations. The antiPD-L1 gene encodes an immunomodulatory protein that activates the immune system to kill tumor cells. The plasmid is activated in high lactic acid environments, and Plldr(new)-antiPD-L1 produces an immunomodulatory drug under these conditions. The plasmid is more sensitive and precise when lactic acid concentration increases and remains unaffected by low oxygen conditions.</p>
+
     <p>
 +
        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.
 +
    </p>
  
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     <!-- Figure 1 -->
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     <div style="text-align:center;">
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    <figure>
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         <img src="https://static.igem.wiki/teams/5526/bba-k5526007-2/1.png" alt="Figure 1. The plasmid map of Plldr(new)-Azurin">
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         <img src="https://static.igem.wiki/teams/5526/bba-k5526007/1.png" alt="Plasmid map of Plldr(new)-anti PD-L1">
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         <div class="caption">Figure 1. The plasmid map of Plldr(new)-Azurin.</div>
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         <figcaption>Figure 1. The plasmid map of Plldr(new)-anti PD-L1</figcaption>
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     </div>
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     </figure>
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     <h3>Engineering Principle</h3>
 
     <h3>Engineering Principle</h3>
−
     <p>We constructed Plldr(new)-antiPD-L1 (BBa_K5526007) by combining Plldr-new, antiPD-L1, and pUC57-mini to create Plldr(new)-antiPD-L1 (plactate2-antiPD-L1).</p>
+
     <p>
 +
        In this plasmid, we combined Plldr-new (BBa_K5526001), Azurin (BBa_K5526000), and pUC57-mini (BBa_K3983004) to create Plldr(new)-Azurin (plactate2-Azurin).
 +
    </p>
  
 
     <h3>Experimental Approach</h3>
 
     <h3>Experimental Approach</h3>
−
     <p>We applied PCR to the genes antiPD-L1 (372bp) and pUC57-plldr-new (3800bp). Agarose gel electrophoresis was used to confirm the PCR results, as shown in Figure 2. The expected lengths of the products were confirmed: pUC57-plldr-new (plactate 2) is 3800bp, and antiPD-L1 is 372bp.</p>
+
     <p>
 +
        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.
 +
    </p>
  
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     <!-- Figure 2 -->
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     <div style="text-align:center;">
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    <figure>
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         <img src="https://static.igem.wiki/teams/5526/bba-k5526007-2/2.jpg" alt="Figure 2. Identification of PCR products by agarose gel electrophoresis">
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         <img src="https://static.igem.wiki/teams/5526/bba-k5526007/2.jpg" alt="PCR production identification by agarose gel electrophoresis">
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         <div class="caption">Figure 2. Identification of PCR products by agarose gel electrophoresis. Left: 3800 bp for pUC57-plldr. Right: 444 bp for p2-Azurin.</div>
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         <figcaption>Figure 2. The identification of PCR production by agarose gel electrophoresis. Left: The graph shows that plactate 2 has a length of 3800bp. Right: The graph shows that p2-antiPD-L1 has a length of 372bp.</figcaption>
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     <p>We used homologous recombination to combine antiPD-L1 with the new Plldr promoter, forming Plldr(new)-antiPD-L1 (plactate2-antiPD-L1). After heat shock transformation of DH5α cells, colonies grew on the Amp+ medium, confirming successful plasmid uptake. Colony PCR further confirmed the presence of the desired plasmid. The sequencing results also verified that the plasmid was correct, as shown in Figure 3.</p>
+
     <p>
 +
        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.
 +
    </p>
  
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     <!-- Figure 3 -->
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     <div style="text-align:center;">
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    <figure>
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         <img src="https://static.igem.wiki/teams/5526/bba-k5526007-2/3.jpg" alt="Figure 3. PCR identification of plactate2-Azurin plasmid">
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         <img src="https://static.igem.wiki/teams/5526/bba-k5526007/3.jpg" alt="PCR identification of plactate2-antiPD-L1 plasmid">
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         <div class="caption">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.</div>
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         <figcaption>Figure 3. PCR identification of plactate2-antiPD-L1 plasmid. A: The length of p2-antiPD-L1 is 372bp. B: The flora growing in a petri dish. C: Sequencing results from the bio company.</figcaption>
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     </figure>
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     <h3>Characterization/Measurement</h3>
 
     <h3>Characterization/Measurement</h3>
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     <p>We used alkaline lysis to extract plasmids (plactate2-Anti-PD-L1) from bacterial cultures. We then transformed the recombinant plasmid into EcN1917 by heat shock. PCR was used to verify the transformation, as shown in Figure 4. The colony PCR results, detected by agarose gel electrophoresis, showed an amplified band of 550bp, consistent with the target band, confirming successful transformation into EcN1917.</p>
+
     <p>
 +
        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.
 +
    </p>
  
−
     <!-- Figure 4 -->
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     <div style="text-align:center;">
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    <figure>
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         <img src="https://static.igem.wiki/teams/5526/bba-k5526007-2/4.jpg" alt="Figure 4. PCR identification of EcN1917 transformants">
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         <img src="https://static.igem.wiki/teams/5526/bba-k5526007/4.jpg" alt="PCR identification of EcN1917 transformants">
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         <div class="caption">Figure 4. PCR identification of EcN1917 transformants. A: Agarose gel showing 550 bp target band. B: Flora growing on a petri dish.</div>
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         <figcaption>Figure 4. PCR identification of EcN1917 transformants. A: PCR production of plactate2-Anti-PD-L1 transformants by agarose gel electrophoresis. B: Flora growing in a petri dish.</figcaption>
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     </div>
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     <p>We cultured bacteria with the plasmids under varying OD values (0.3, 0.6, 0.8, 1.0) and lactic acid concentrations (0mM, 2mM, 5mM, 10mM). Using a nanodrop, we measured protein concentration and concluded that the highest protein concentration occurred when OD600 was 0.6 and lactic acid concentration was 5mM. SDS-PAGE was used to verify the obtained proteins.</p>
+
     <p>
 +
        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.
 +
    </p>
  
−
     <!-- Figure 5 -->
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    <figure>
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         <img src="https://static.igem.wiki/teams/5526/bba-k5526007-2/5.jpg" alt="Figure 5. Effects of bacterial concentration and lactic acid on plactate2-Azurin expression">
−
         <img src="https://static.igem.wiki/teams/5526/bba-k5526007/5.jpg" alt="Effects of bacterial concentration and lactic acid concentration on protein expression">
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         <div class="caption">Figure 5. Effects of bacterial concentration and lactic acid on plactate2-Azurin expression.</div>
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         <figcaption>Figure 5. The effects of bacterial concentration and lactic acid concentration on the expression of plactate2-antiPD-L1.</figcaption>
+
     </div>
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     </figure>
+
  
 
     <h3>Other Tests</h3>
 
     <h3>Other Tests</h3>
−
     <p>We expanded the culture and induced protein expression using 5mM lactic acid in EcN1917. The experimental results, shown in Figure 6, confirmed that the Anti-PD-L1 protein was successfully expressed. The protein size was approximately 24kDa, consistent with the expected result. Future experiments will study the expression and tumor inhibition effect of the constructed probiotics in a tumor environment.</p>
+
     <p>
 +
        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).
 +
    </p>
  
−
     <!-- Figure 6 -->
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     <div style="text-align:center;">
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    <figure>
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         <img src="https://static.igem.wiki/teams/5526/bba-k5526007-2/6.jpg" alt="Figure 6. Detection of Azurin protein expression by SDS-PAGE">
−
         <img src="https://static.igem.wiki/teams/5526/bba-k5526007/6.jpg" alt="Detection of Anti-PD-L1 protein expression by SDS-PAGE">
+
         <div class="caption">Figure 6. Detection of Azurin protein expression by SDS-PAGE.</div>
−
         <figcaption>Figure 6. Detection of Anti-PD-L1 protein expression by SDS-PAGE.</figcaption>
+
     </div>
−
     </figure>
+
  
 
     <h3>Summary</h3>
 
     <h3>Summary</h3>
−
     <p>EcN drug molecular delivery carriers offer good compliance, long-lasting efficacy, and therapeutic precision. However, several challenges remain before they can be applied clinically. Future animal experiments will focus on verifying the tumor inhibition effects of engineered probiotics. Additional research is necessary to improve EcN strains for safe and effective use as probiotic drugs. Much work remains to achieve precision tumor treatment using EcN drug delivery vectors.</p>
+
     <p>
−
 
+
        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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Revision as of 14:48, 30 September 2024


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.