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| | ===Characterization=== | | ===Characterization=== |
| − | In order to obtain proteins, test suitable expression conditions, and evaluate the function of TRn4-mfp5, we chose three different expression vectors (Fig. 3)—pET-28a(+), pET-SUMO, and pET-21a(+)—and tried different strategies for TRn4-mfp5 protein production and purification. | + | In order to obtain proteins, test suitable expression conditions, and evaluate the function of TRn4-mfp5, we chose three different expression vectors (Fig. 3)—pET-28a(+), pET SUMO, and pET-21a(+)—and tried different strategies for TRn4-mfp5 protein production and purification. |
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| | <div class="module"> | | <div class="module"> |
| | <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/three-plasmid-trn4mfp5.webp" width="700" height="auto" alt="Protein purification"> | | <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/three-plasmid-trn4mfp5.webp" width="700" height="auto" alt="Protein purification"> |
| − | <p><b>Fig. 1 | Three different vectors used in protein expression.</b></p> | + | <p><b>Fig. 3 | Three different vectors used in protein expression.</b></p> |
| | <p><b>a.</b> The plasmid map of pET-28a(+)-His-SUMO-TRn4-mfp5; | | <p><b>a.</b> The plasmid map of pET-28a(+)-His-SUMO-TRn4-mfp5; |
| − | <b>b.</b> The plasmid map of pET-SUMO-TRn4-mfp5; | + | <b>b.</b> The plasmid map of pET SUMO-TRn4-mfp5; |
| | <b>c.</b> The plasmid map of pET-21a(+)-TRn4-mfp5.</p> | | <b>c.</b> The plasmid map of pET-21a(+)-TRn4-mfp5.</p> |
| | </div> | | </div> |
| − | </body>
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| − | </html>
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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">
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| − | <style>
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| − | .module {
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| − | border: 1px solid #ccc; /* 边框 */
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| − | padding: 20px; /* 内边距 */
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| − | margin: 20px auto; /* 外边距，自动居中 */
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| − | width: 800px; /* 模块宽度 */
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| − | text-align: center; /* 内容居中 */
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| − | box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */
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| − | font-size: 16px; /* 设置字体大小 */
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| − | line-height: 1.6; /* 设置行高，使文本更易读 */
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| − | }
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| − |
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| − | /* 为图片说明文本设定特定的样式 */
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| − | .module p {
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| − | font-size: 16px; /* 确保所有段落的字体大小一致 */
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| − | line-height: 1.6; /* 调整行距 */
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| − | }
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| − | </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/trn4-mfp5/16-37-lb-pet21a.webp" width="700" height="auto" alt="Protein purification">
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| − | <p><b>Fig. 2 | Comparison of fusion protein expression in different temperature use vector pET-21a(+).</b></p>
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| − | <p>
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| − | Lanes 1-6 (LB 37°C 4 h):
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| − | 1. Protein ladder;
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| − | 2. total liquid (+IPTG);
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| − | 3. supernatant (+IPTG);
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| − | 4. precipitate (+IPTG);
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| − | 5. total liquid (-IPTG);
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| − | 6. supernatant (-IPTG);
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| − | 7. precipitate (-IPTG);
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| − | Lanes 8-13 (TB 16°C 20 h):
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| − | 8. Protein ladder;
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| − | 9. total liquid (+IPTG);
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| − | 10. supernatant (+IPTG);
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| − | 11. precipitate (+IPTG);
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| − | 12. total liquid (-IPTG);
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| − | 13. supernatant (-IPTG);
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| − | 14. precipitate (-IPTG).
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| − | </p>
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| − | </div>
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| − |
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| − | <div class="module">
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| − | <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/tb-lb-prt21a.webp" width="700" height="auto" alt="Protein purification">
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| − | <p><b>Fig. 3 | Comparison of fusion protein expression in LB and TB media use vector pET-21a(+).</b></p>
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| − | <p>
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| − | 1. Protein ladder;
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| − | Lanes 2-7 (LB 16°C 20 h):
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| − | 2. total liquid (+IPTG);
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| − | 3. supernatant (+IPTG);
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| − | 4. precipitate (+IPTG);
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| − | 5. total liquid (-IPTG);
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| − | 6. supernatant (-IPTG);
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| − | 7. precipitate (-IPTG);
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| − |
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| − | Lanes 8-13 (TB 16°C 20 h):
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| − | 8. Protein ladder;
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| − | 9. total liquid (+IPTG);
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| − | 10. supernatant (+IPTG);
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| − | 11. precipitate (+IPTG);
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| − | 12. total liquid (-IPTG);
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| − | 13. supernatant (-IPTG);
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| − | 14. precipitate (-IPTG).
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| − | </p>
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| − | </div>
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| − |
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| − | <div class="module">
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| − | <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/rostta-bl21-de3-trn4-mfp5png.webp" width="700" height="auto" alt="Protein purification">
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| − | <p><b>Fig. 4 | Comparison of fusion protein expression in <i>E. coli</i> strains BL21(DE3) and Rosetta.</b></p>
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| − | <p>
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| − | 1. Protein ladder;
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| − | Lanes 2-4 (BL21(DE3) LB 37℃ 4h)
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| − | 2. total liquid (+IPTG);
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| − | 3. supernatant (+IPTG);
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| − | 4. precipitate (+IPTG);
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| − | Lanes 5-7 (Rosetta LB 37℃ 4h)
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| − | 5. total liquid (+IPTG);
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| − | 6. supernatant (+IPTG);
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| − | 7. precipitate (+IPTG).
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| − | </p>
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| − | </div>
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| − | </body>
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| − | </html>
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| − |
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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">
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| − | <style>
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| − | .module {
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| − | border: 1px solid #ccc; /* 边框 */
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| − | padding: 20px; /* 内边距 */
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| − | margin: 20px auto; /* 外边距，自动居中 */
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| − | width: 800px; /* 模块宽度 */
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| − | text-align: center; /* 内容居中 */
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| − | box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */
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| − | font-size: 16px; /* 设置字体大小 */
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| − | line-height: 1.6; /* 设置行高，使文本更易读 */
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| − | }
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| − |
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| − | .module p {
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| − | font-size: 16px; /* 确保所有段落的字体大小一致 */
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| − | line-height: 1.6; /* 调整行距 */
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| − | }
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| − | </style>
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| − | </head>
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| − | <body>
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| − | <div class="module">
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| − | <p>After considering both expression efficiency and practical experimental constraints, we decided to express the fusion protein at 37°C for 4 h in LB medium using the pET-SUMO-TRn4-mfp5 plasmid.</p>
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| − | <p>As shown in Figures 4-6, the target protein was present in the pellet after cell lysis. Therefore, we denatured the pellet of the fusion protein TRn4-mfp5 with 8M urea overnight and renatured it through dialysis. This process resulted in some protein loss, as confirmed by SDS-PAGE analysis.</p>
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| − | <p>Consequently, we proceeded to purify the fusion protein TRn4-mfp5 using a Ni-NTA Gravity Column.</p>
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| − | <p>The target protein bands were present in lanes 4 to 7, indicating successful expression of the target protein, with a particularly strong band in the supernatant after denaturation (Fig. 7, lane 7). After purification, the target protein was mainly found in the 150 mM and 300 mM imidazole elution fractions.</p>
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| − | </div>
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| − |
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| − | <div class="module">
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| − | <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/purification-trn4-mfp5.webp" width="700" height="auto" alt="Protein purification">
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| − | <p><b>Fig. 5 | SDS-PAGE of purified fusion protein TRn4-mfp5(35.4 kDa) uses vector pET-SUMO.</b></p>
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| − | <p>
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| − | Lane 1: Protein - Binding buffer;<br>
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| − | Lane 2: 20 mM imidazole and 8 M urea elution;<br>
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| − | Lane 3: 50 mM imidazole and 8 M urea elution;<br>
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| − | Lane 4: 150 mM imidazole and 8 M urea elution;<br>
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| − | Lane 5: 300 mM imidazole and 8 M urea elution;<br>
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| − | Lane 6: 500 mM imidazole and 8 M urea elution;<br>
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| − | Lane 7: Supernatant;<br>
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| − | Lane 8: Impurities;<br>
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| − | Lane 9: Protein ladder.
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| − | </p>
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| − | </div>
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| − |
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| − | <div class="module">
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| − | <p>To further confirm the expression of TRn4-mfp5, we performed a Western blot, which provided a clear and definitive conclusion, verifying the successful expression of the TRn4-mfp5 protein under the conditions mentioned above.</p>
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| − | </div>
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| − |
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| − | <div class="module">
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| − | <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/wb-all-final.webp" width="700" height="auto" alt="Protein purification">
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| − | <p><b>Fig. 6 | Western Blot of purified fusion protein TRn4-mfp5(35.4 kDa) uses vector pET-SUMO.</b></p>
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| − | <p>
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| − | <b>a.</b> Western blot of the pre-expressed protein;<br>
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| − | <b>b.</b> Western blot after column purification of the supernatant following denaturation.
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| − | </p>
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| − | </div>
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| − | </body>
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| − | </html>
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| − |
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| − | ====Adhesive test====
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| − | We obtained protein samples of TRn4-mfp5 by freezedrying 24 h (Fig. 9). The final yield was about 25 mg/L bacterial culture.
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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">
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| − | <style>
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| − | .module {
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| − | border: 1px solid #ccc; /* 边框 */
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| − | padding: 20px; /* 内边距 */
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| − | margin: 20px auto; /* 外边距，自动居中 */
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| − | width: 800px; /* 模块宽度 */
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| − | text-align: center; /* 内容居中 */
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| − | box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */
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| − | }
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| − | </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/trn4-mfp5/protein-freeze-actual-picture-new.webp" width="700" height="auto" alt="Protein purification">
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| − | <p><b>Fig. 7 | The protein sample freeze-dried by a lyophilizer.</b></p>
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| − | </div>
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| − | </body>
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| − | </html>
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| − |
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| − | Next, we dissolved protein samples in Buffer A (10 mL 20 mM Tris pH8) to reach 0.3 mg/mL, and conduct adhesive ability tests on the fusion protein(Fig. 10). 200 μL of the protein solution was applied, and the pipette tip was placed on a plastic Petri dish lid. After incubation at 37°C for 8 h, the pipette tip successfully adhered.
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| − |
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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">
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| − | <style>
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| − | .module {
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| − | border: 1px solid #ccc; /* 边框 */
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| − | padding: 20px; /* 内边距 */
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| − | margin: 20px auto; /* 外边距，自动居中 */
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| − | width: 800px; /* 模块宽度 */
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| − | text-align: center; /* 内容居中 */
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| − | box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */
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| − | }
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| − | </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/trn4-mfp5/part-fig10.webp" width="700" height="auto" alt="Protein purification">
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| − | <p><b>Fig. 8 | Adhesive ability test of fusion protein on plastic surface</b></p>
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| − | </div>
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| − | </body>
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| − | </html>
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| − | <html lang="en">
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| − | <meta charset="UTF-8">
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| − | <title>Viscosity Calculations</title>
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| − | <style>
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| − | body {
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| − | font-family: Arial, sans-serif;
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| − | }
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| − | .calculation {
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| − | p {
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| − | font-size: 1.2em;
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| − | }
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| − | <b>Viscosity Calculations</b>
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| − |
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| − | <p>Surface Area Calculation:</p>
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| − | <p>The surface area of a 10 µl pipette tip with an inner diameter of 3.7 mm is calculated as:</p>
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| − | <div class="calculation">
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| − | <p>S = π × r² = π × (0.185 cm)² = 0.1075 cm²</p>
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| − | </div>
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| − |
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| − | <p>Force Calculation:</p>
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| − | <p>The total mass is (5.951 + 0.448 × 3) grams, and the force is:</p>
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| − | <div class="calculation">
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| − | <p>F = 7.295 g × 9.8 N/kg = 0.07149 N</p>
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| − | </div>
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| − |
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| − | <p>Adhesive Force Calculation:</p>
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| − | <p>The adhesive force produced by the protein is:</p>
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| − | <div class="calculation">
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| − | <p>P = F / S = 0.07149 N / 0.1075 cm² = 0.665 N/cm² ≈ 6.65 KPa</p>
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| − | </div>
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| − |
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| − | <p>Adhesive Force per Milligram of Protein:</p>
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| − | <p>The adhesive force per milligram of protein is:</p>
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| − | <div class="calculation">
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| − | <p>P' = P / m = 6.65 KPa / 1 mg = 6.65 KPa/mg</p>
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| − | </div>
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| − |
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| | </body> | | </body> |
| | </html> | | </html> |
In order to obtain proteins with adhesive properties, we used the pET-SUMO vector to express TRn4-mfp5 ( BBa_K5398020) ). We tried different strategies for TRn4-mfp5 protein production and purification and tested its function.
In order to obtain proteins, test suitable expression conditions, and evaluate the function of TRn4-mfp5, we chose three different expression vectors (Fig. 3)—pET-28a(+), pET SUMO, and pET-21a(+)—and tried different strategies for TRn4-mfp5 protein production and purification.
[1] Jung H., Pena-Francesch A., Saadat A, et al. Molecular tandem repeat strategy for elucidating mechanical properties of high-strength proteins[J]. PNAS, 2016, 113(23), 6478–6483.
[2] Zhang C, Wu B, Zhou Y, et al. Mussel-inspired hydrogels: from design principles to promising applications[J]. Chem Soc Rev, 2020, 49(3605): 3605-3637.
