Difference between revisions of "Part:BBa K5398005"
| Line 5: | Line 5: | ||
<p>In order to obtain proteins with self-healing properties, we used the pET29a(+) vector to express TRn5 (BBa_K5398001) (Fig. 1). We tried different strategies for TRn5 protein production and purification and tested its function. </p> | <p>In order to obtain proteins with self-healing properties, we used the pET29a(+) vector to express TRn5 (BBa_K5398001) (Fig. 1). We tried different strategies for TRn5 protein production and purification and tested its function. </p> | ||
| − | |||
| − | |||
| − | |||
| − | + | ====Protein expression==== | |
| − | + | ||
| − | <p>We expressed the protein in <i>E.coli</i> BL21 (DE3) using LB medium. After incubation at 37℃ for | + | <p>We expressed the protein in <i>E.coli</i> BL21 (DE3) using LB medium. After incubation at 37℃ for 5 h and 30℃ for 9 h, respectively, we found that most TRn5 (17.58 kDa) existed in precipitation and the TRn5 expression level at two temperatures had little difference (Fig. 1).</p> |
| − | <html> | + | <html lang="zh"> |
| − | <center><img src="https://static.igem.wiki/teams/5398/trn5/sds-page-1- | + | <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: 700px; /* 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"> | ||
| + | <img src="https://static.igem.wiki/teams/5398/trn5/sds-page-1-2.webp" width="600" height="auto" alt="Protein purification"> | ||
| + | <p><b>Fig. 1 SDS-PAGE of expression products of TRn5.</b></p> | ||
| + | </div> | ||
| + | </body> | ||
</html> | </html> | ||
| − | <p | + | <p>Then, we purified TRn5 by Immobilized Metal Affinity Chromatography (IMAC). However, the TRn5 expression level was too low to verify by SDS-PAGE (Fig. 2).</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. 2 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> | </html> | ||
| − | |||
| − | |||
| − | <p>To optimize the TRn5 expression, we reviewed plenty of literature, from which we found that TRn5 could easily be dissolved in 5% acetic acid (pH≈3) due to the existence of Histidine. Thus, we used a new protocol to obtain the purified TRn5. Solubilized in 5% acetic acid, the band of TRn5 was seen clearly, which means success of this purification manner (Fig. | + | <p>To optimize the TRn5 expression, we reviewed plenty of literature, from which we found that TRn5 could easily be dissolved in 5% acetic acid (pH≈3) due to the existence of Histidine. Thus, we used a new protocol to obtain the purified TRn5. Solubilized in 5% acetic acid, the band of TRn5 was seen clearly, which means success of this purification manner (Fig. 3).</p> |
| − | <html> | + | <html lang="zh"> |
| − | <center><img src="https://static.igem.wiki/teams/5398/trn5/sds-page-3-1.webp" | + | <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. 3 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> | </html> | ||
| − | + | ====Protein self-healing==== | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | ==== | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| Line 57: | Line 98: | ||
<partinfo>BBa_K5398005 SequenceAndFeatures</partinfo> | <partinfo>BBa_K5398005 SequenceAndFeatures</partinfo> | ||
| + | ===Reference === | ||
| + | <p>[1] JUNG H, PENA-FRANCESCH A, SAADAT A, et al. Molecular tandem repeat strategy for elucidating mechanical properties of high-strength proteins[J].<i> PNAS</i>, 2016, 113(23): 6478-6483.</p> | ||
| + | <p>[2] PENA-FRANCESCH A, JUNG H, DEMIREL M C, et al. Biosynthetic self-healing materials for soft machines [J]. <i>Nat. Mater.</i>, 2020, 19(11): 1230-1235.</p> | ||
| + | <p>[3] PENA-FRANCESCH A, FLOREZ S, JUNG H, et al. Materials Fabrication from Native and Recombinant Thermoplastic Squid Proteins[J].<i> Adv. Funct.</i>, 2014, 24(47): 7401-7409.</p> | ||
| + | <p>[4] GUERETTE P A, HOON S, SEOW Y, et al. Accelerating the design of biomimetic materials by integrating RNA-seq with proteomics and materials science[J]. <i>Nat. Biotechnol.</i>, 2013, 31(10): 908-915.</p> | ||
| + | <p>[5] DING D, GUERETTE P A, HOON S, et al. Biomimetic Production of Silk-Like Recombinant Squid Sucker Ring Teeth Proteins[J]. <i>Biomacromolecules</i>, 2014, 15(9): 3278-3289.</p> | ||
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
Revision as of 14:48, 17 September 2024
pET29a(+)-TRn5
In order to obtain proteins with self-healing properties, we used the pET29a(+) vector to express TRn5 (BBa_K5398001) (Fig. 1). We tried different strategies for TRn5 protein production and purification and tested its function.
Protein expression
We expressed the protein in E.coli BL21 (DE3) using LB medium. After incubation at 37℃ for 5 h and 30℃ for 9 h, respectively, we found that most TRn5 (17.58 kDa) existed in precipitation and the TRn5 expression level at two temperatures had little difference (Fig. 1).
Fig. 1 SDS-PAGE of expression products of TRn5.
Then, we purified TRn5 by Immobilized Metal Affinity Chromatography (IMAC). However, the TRn5 expression level was too low to verify by SDS-PAGE (Fig. 2).
Fig. 2 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.
To optimize the TRn5 expression, we reviewed plenty of literature, from which we found that TRn5 could easily be dissolved in 5% acetic acid (pH≈3) due to the existence of Histidine. Thus, we used a new protocol to obtain the purified TRn5. Solubilized in 5% acetic acid, the band of TRn5 was seen clearly, which means success of this purification manner (Fig. 3).
Fig. 3 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.
Protein self-healing
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Reference
[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] PENA-FRANCESCH A, JUNG H, DEMIREL M C, et al. Biosynthetic self-healing materials for soft machines [J]. Nat. Mater., 2020, 19(11): 1230-1235.
[3] PENA-FRANCESCH A, FLOREZ S, JUNG H, et al. Materials Fabrication from Native and Recombinant Thermoplastic Squid Proteins[J]. Adv. Funct., 2014, 24(47): 7401-7409.
[4] GUERETTE P A, HOON S, SEOW Y, et al. Accelerating the design of biomimetic materials by integrating RNA-seq with proteomics and materials science[J]. Nat. Biotechnol., 2013, 31(10): 908-915.
[5] DING D, GUERETTE P A, HOON S, et al. Biomimetic Production of Silk-Like Recombinant Squid Sucker Ring Teeth Proteins[J]. Biomacromolecules, 2014, 15(9): 3278-3289.