Difference between revisions of "Part:BBa K5398001:Experience"

 
(Applications of BBa_K5398001)
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===Applications of BBa_K5398001===
 
===Applications of BBa_K5398001===
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<html>
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<p>In order to obtain proteins with self-healing properties, we used  the pET-29a(+) vector to express TRn5 (<a href="https://parts.igem.org/Part:BBa_K5398001"> BBa_K5398001)</a> ). We tried different strategies for TRn5 protein production and purification and tested its function. </p>
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</html>
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 +
__TOC__
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===Characterization===
 +
====Cloning strategy and results====
 +
<p>In our project, we constructed pET-29a(+)-TRn5 plasmid to express TRn5 by inserting the coding sequences (CDS) of TRn5 into pET-29a(+) vector by the following steps.(Fig. 1a). </p>
 +
 +
<ul>
 +
    <li>PCR amplification of TRn5 and pET-29a(+) vector respectively. This PCR produces the pET-29a(+)-TRn5 part ready for In-fusion Cloning (Fig. 1b,c).</li>
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<html lang="zh">
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<head>
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    <meta charset="UTF-8">
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    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <style>
 +
        .module {
 +
            border: 1px solid #ccc; /* 边框 */
 +
            padding: 20px; /* 内边距 */
 +
            margin: 20px auto; /* 外边距,自动居中 */
 +
            width: 800px; /* 模块宽度 */
 +
            text-align: center; /* 内容居中 */
 +
            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */
 +
        }
 +
    </style>
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</head>
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<body>
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    <div class="module">
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        <img src="https://static.igem.wiki/teams/5398/trn5/map-and-jiaotu-of-trn5-2.webp" width="800" height="auto" alt="Protein purification">
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        <p><b>Fig. 1 | The plasmid map of pET-29a(+)-TRn5 and 1% agarose gel electrophoresis of the PCR amplified pET-29a(+)-TRn5 parts.</b></p>
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      <p><b>a.</b> The plasmid map of pET-29a(+)-TRn5;
 +
      <b>b.</b> 1% agarose gel electrophoresis of the PCR amplified TRn5 (K5398001) (527 bp);
 +
      <b>c.</b> 1% agarose gel electrophoresis of the PCR amplified pET-29a(+) vector (5170 bp).</p>
 +
    </div>
 +
</body>
 +
</html>
 +
 +
    <li>In-fusion Cloning of purified PCR amplified TRn5 and the pET-29a(+) vector parts for the efficient construction of the TRn5 coding sequence under the transcriptional control of the T7lac promoter. The recombinant plasmid was transferred into <i>E.coli</i> DH5α.</li>
 +
    <li>Verification of target recombinant plamid. Colony PCR was used to screen for clones with inserts of the desired sizes and Sanger sequencing confirmed the lengths and compositions of the clones after plasmid isolation, from which we concluded that the pET-29a(+)-TRn5 was conducted plasmid successfully (Fig. 2).</li>
 +
 +
<html lang="zh">
 +
<head>
 +
    <meta charset="UTF-8">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <style>
 +
        .module {
 +
            border: 1px solid #ccc; /* 边框 /
 +
*            padding: 20px; /* 内边距 /
 +
*            margin: 20px auto; /* 外边距,自动居中 /
 +
*            width: 500px; /* 模块宽度 /
 +
*            text-align: center; /* 内容居中 /
 +
*            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */
 +
        }
 +
    </style>
 +
</head>
 +
<body>
 +
    <div class="module">
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        <img src="https://static.igem.wiki/teams/5398/trn5/p-1.webp" width="800" height="auto" alt="Protein purification">
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        <p><b>Fig. 2 | Verification of recombinant plamid pET-29a(+)-TRn5.</b></p>
 +
      <p><b>a.</b> 1% agarose gel electrophoresis of colony PCR of  using T7 and T7 ter primes;
 +
      <b>b.</b> The result of sequencing the TRn5 of the recombinant plamid.
 +
    </div>
 +
</body>
 +
</html>
 +
 +
====Protein expression====
 +
<p>We expressed the protein in <i>E.coli</i> BL21 (DE3) using LB medium. After incubation at 23℃ for 16 h and 37℃ for 5 h respectively, we found that most TRn5 (17.58 kDa) existed in precipitate as stated in previous research and the TRn5 expression level at two temperatures had little difference (Fig. 3).</p>
 +
 +
<html lang="zh">
 +
<head>
 +
    <meta charset="UTF-8">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <title>模块示例</title>
 +
    <style>
 +
        .module {
 +
-            border: 1px solid #ccc; /
 +
-            padding: 20px; /
 +
-            margin: 20px auto; /
 +
-            width: 500px; /
 +
-            text-align: center; /
 +
-            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /
 +
      }
 +
  </style>
 +
</head>
 +
<body>
 +
    <div class="module">
 +
        <img src="https://static.igem.wiki/teams/5398/trn5/sds-page-1-2.webp" width="600" height="auto" alt="Protein purification">
 +
        <p><b>Fig. 3 | SDS-PAGE of expression products of TRn5.</b></p>
 +
    <p>Lane 1: marker; lanes 2 to 4: whole-cell lysate, supernatant and pellet from uninduced cells at 23℃, respectively; lanes 5 to 7: whole-cell lysate, supernatant and pellet from induced cells at 23℃, respectively.  lanes 8 to 10: whole-cell lysate, supernatant and pellet from uninduced cells at 37℃, respectively; lanes 11 to 13: whole-cell lysate, supernatant and pellet from induced cells at 37℃, respectively. </p>
 +
    </div>
 +
</body>
 +
</html>
 +
 +
<p>Then, we denatured TRn5 with 8 mM urea overnight and renatured it by dialysis, which proved great protein losses as shown in SDS-PAGE. As a result, when we purified TRn5 by Immobilized Metal Affinity Chromatography (IMAC), the TRn5 expression level was too low to verify (Fig. 4).</p>
 +
 +
<html lang="zh">
 +
<head>
 +
    <meta charset="UTF-8">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <title>模块示例</title>
 +
    <style>
 +
        .module {
 +
-            border: 1px solid #ccc; /
 +
-            padding: 20px; /
 +
-            margin: 20px auto; /
 +
-            width: 500px; /
 +
-            text-align: center; /
 +
-            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /
 +
      }
 +
  </style>
 +
</head>
 +
<body>
 +
    <div class="module">
 +
        <img src="https://static.igem.wiki/teams/5398/trn5/sds-page-2-1.webp" width="600" height="auto" alt="Protein purification">
 +
        <p><b>Fig. 4 | SDS-PAGE of expression products of TRn5 purified by IMAC.</b></p>
 +
    <p>Lane 1: marker; lanes 2 to 11, induced cell sample at 23℃; lane 2: pellet; lane 3: sample washed with denaturing buffer with 8 mM urea; lane 4: sample after dialysis overnight; lane 5: sample after being bound to Ni-NTA resin; lane 6: sample eluted with 20 mM Tris-HCl; lane 7: sample eluted with 20 mM imidazole;  lane 8: sample eluted with 50 mM imidazole; lane 9: sample eluted with 150 mM imidazole; lane 10: sample eluted with 300 mM imidazole; lane 11: sample eluted with 500 mM imidazole.</p>
 +
    </div>
 +
</body>
 +
</html>
 +
 +
<p>In order to optimize the expression of TRn5, we conducted a comprehensive review of the existing literature, revealing that the presence of Histidine facilitates the effortless dissolution of TRn5 in 5% acetic acid. Consequently, we implemented a novel protocol for the purification of TRn5. Upon solubilization in 5% acetic acid, a distinct and clear band of TRn5 was observed (Fig. 5).</p>
 +
 +
<html lang="zh">
 +
<head>
 +
    <meta charset="UTF-8">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <title>模块示例</title>
 +
    <style>
 +
        .module {
 +
-            border: 1px solid #ccc; /
 +
-            padding: 20px; /
 +
-            margin: 20px auto; /
 +
-            width: 500px; /
 +
-            text-align: center; /
 +
-            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /
 +
      }
 +
  </style>
 +
</head>
 +
<body>
 +
    <div class="module">
 +
        <img src="https://static.igem.wiki/teams/5398/trn5/sds-page-3-1.webp" width="375" height="auto" alt="Protein purification">
 +
        <p><b>Fig. 5 | SDS-PAGE of expression products of TRn5 using a new protocol.</b></p>
 +
    <p>Lane 1: marker; lanes 2 to 4: whole-cell lysate, supernatant and pellet from induced cells at 37℃, respectively; lane 5: sample washed with 5% acetic acid.</p>
 +
    </div>
 +
</body>
 +
</html>
 +
 +
====Self-healing test====
 +
<p>We obtained protein samples of TRn5 by freezedrying 24 h (Fig. 6). The final yield was about 15 mg/1 L bacterial culture. Next, we dissolved protein samples in 5% acetic acid to reach 20 mg/μL, cast them into square models and dried them at 70℃ for 3 h to obtain protein films.</p>
 +
 +
<html lang="zh">
 +
<head>
 +
    <meta charset="UTF-8">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <title>模块示例</title>
 +
    <style>
 +
        .module {
 +
-            border: 1px solid #ccc; /
 +
-            padding: 20px; /
 +
-            margin: 20px auto; /
 +
-            width: 500px; /
 +
-            text-align: center; /
 +
-            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /
 +
      }
 +
  </style>
 +
</head>
 +
<body>
 +
    <div class="module">
 +
        <img src="https://static.igem.wiki/teams/5398/trn5/freezedrying.webp" width="500" height="auto" alt="Protein purification">
 +
        <p><b>Fig. 6 | The protein sample freeze-dried by a lyophilizer.</b></p>
 +
    </div>
 +
</body>
 +
</html>
 +
 +
<p>To examine the property of self-healing of TRn5, we punctured a TRn5 protein film to create a hole defect by a needle (Fig. 7a). After putting the punctured film at room temperature for 12 h, we clearly saw the hole defect healing (Fig. 7b).So it was proved that TRn5 has a self-healing property.</p>
 +
 +
<html lang="zh">
 +
<head>
 +
    <meta charset="UTF-8">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <title>模块示例</title>
 +
    <style>
 +
        .module {
 +
-            border: 1px solid #ccc; /
 +
-            padding: 20px; /
 +
-            margin: 20px auto; /
 +
-            width: 500px; /
 +
-            text-align: center; /
 +
-            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /
 +
      }
 +
  </style>
 +
</head>
 +
<body>
 +
    <div class="module">
 +
        <img src="https://static.igem.wiki/teams/5398/trn5/self-healing-of-trn5-protein-films.webp" width="600" height="auto" alt="Protein purification">
 +
        <p><b>Fig. 7 | Self-healing of TRn5 protein films after puncture damage.</b></p>
 +
    <p><b>a.</b> A hole defect was left by a needle through the film; <b>b.</b> Puncture damage was healed.</p>
 +
    </div>
 +
</body>
 +
</html>
 +
 +
<html>
 +
<p>More information about the project for which the part was created:<a href="https://2024.igem.wiki/nau-china/description"> SAMUS (NAU-CHINA 2024).</a></p>
 +
</html>
  
 
===User Reviews===
 
===User Reviews===

Revision as of 00:41, 27 September 2024


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K5398001

In order to obtain proteins with self-healing properties, we used the pET-29a(+) vector to express TRn5 ( BBa_K5398001) ). We tried different strategies for TRn5 protein production and purification and tested its function.

Characterization

Cloning strategy and results

In our project, we constructed pET-29a(+)-TRn5 plasmid to express TRn5 by inserting the coding sequences (CDS) of TRn5 into pET-29a(+) vector by the following steps.(Fig. 1a).

  • PCR amplification of TRn5 and pET-29a(+) vector respectively. This PCR produces the pET-29a(+)-TRn5 part ready for In-fusion Cloning (Fig. 1b,c).
  • Protein purification

    Fig. 1 | The plasmid map of pET-29a(+)-TRn5 and 1% agarose gel electrophoresis of the PCR amplified pET-29a(+)-TRn5 parts.

    a. The plasmid map of pET-29a(+)-TRn5; b. 1% agarose gel electrophoresis of the PCR amplified TRn5 (K5398001) (527 bp); c. 1% agarose gel electrophoresis of the PCR amplified pET-29a(+) vector (5170 bp).

  • In-fusion Cloning of purified PCR amplified TRn5 and the pET-29a(+) vector parts for the efficient construction of the TRn5 coding sequence under the transcriptional control of the T7lac promoter. The recombinant plasmid was transferred into E.coli DH5α.
  • Verification of target recombinant plamid. Colony PCR was used to screen for clones with inserts of the desired sizes and Sanger sequencing confirmed the lengths and compositions of the clones after plasmid isolation, from which we concluded that the pET-29a(+)-TRn5 was conducted plasmid successfully (Fig. 2).
  • Protein purification

    Fig. 2 | Verification of recombinant plamid pET-29a(+)-TRn5.

    a. 1% agarose gel electrophoresis of colony PCR of using T7 and T7 ter primes; b. The result of sequencing the TRn5 of the recombinant plamid.

    Protein expression

    We expressed the protein in E.coli BL21 (DE3) using LB medium. After incubation at 23℃ for 16 h and 37℃ for 5 h respectively, we found that most TRn5 (17.58 kDa) existed in precipitate as stated in previous research and the TRn5 expression level at two temperatures had little difference (Fig. 3).

    模块示例

    Protein purification

    Fig. 3 | SDS-PAGE of expression products of TRn5.

    Lane 1: marker; lanes 2 to 4: whole-cell lysate, supernatant and pellet from uninduced cells at 23℃, respectively; lanes 5 to 7: whole-cell lysate, supernatant and pellet from induced cells at 23℃, respectively. lanes 8 to 10: whole-cell lysate, supernatant and pellet from uninduced cells at 37℃, respectively; lanes 11 to 13: whole-cell lysate, supernatant and pellet from induced cells at 37℃, respectively.

    Then, we denatured TRn5 with 8 mM urea overnight and renatured it by dialysis, which proved great protein losses as shown in SDS-PAGE. As a result, when we purified TRn5 by Immobilized Metal Affinity Chromatography (IMAC), the TRn5 expression level was too low to verify (Fig. 4).

    模块示例

    Protein purification

    Fig. 4 | SDS-PAGE of expression products of TRn5 purified by IMAC.

    Lane 1: marker; lanes 2 to 11, induced cell sample at 23℃; lane 2: pellet; lane 3: sample washed with denaturing buffer with 8 mM urea; lane 4: sample after dialysis overnight; lane 5: sample after being bound to Ni-NTA resin; lane 6: sample eluted with 20 mM Tris-HCl; lane 7: sample eluted with 20 mM imidazole; lane 8: sample eluted with 50 mM imidazole; lane 9: sample eluted with 150 mM imidazole; lane 10: sample eluted with 300 mM imidazole; lane 11: sample eluted with 500 mM imidazole.

    In order to optimize the expression of TRn5, we conducted a comprehensive review of the existing literature, revealing that the presence of Histidine facilitates the effortless dissolution of TRn5 in 5% acetic acid. Consequently, we implemented a novel protocol for the purification of TRn5. Upon solubilization in 5% acetic acid, a distinct and clear band of TRn5 was observed (Fig. 5).

    模块示例

    Protein purification

    Fig. 5 | SDS-PAGE of expression products of TRn5 using a new protocol.

    Lane 1: marker; lanes 2 to 4: whole-cell lysate, supernatant and pellet from induced cells at 37℃, respectively; lane 5: sample washed with 5% acetic acid.

    Self-healing test

    We obtained protein samples of TRn5 by freezedrying 24 h (Fig. 6). The final yield was about 15 mg/1 L bacterial culture. Next, we dissolved protein samples in 5% acetic acid to reach 20 mg/μL, cast them into square models and dried them at 70℃ for 3 h to obtain protein films.

    模块示例

    Protein purification

    Fig. 6 | The protein sample freeze-dried by a lyophilizer.

    To examine the property of self-healing of TRn5, we punctured a TRn5 protein film to create a hole defect by a needle (Fig. 7a). After putting the punctured film at room temperature for 12 h, we clearly saw the hole defect healing (Fig. 7b).So it was proved that TRn5 has a self-healing property.

    模块示例

    Protein purification

    Fig. 7 | Self-healing of TRn5 protein films after puncture damage.

    a. A hole defect was left by a needle through the film; b. Puncture damage was healed.

    More information about the project for which the part was created: SAMUS (NAU-CHINA 2024).

    User Reviews

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