Difference between revisions of "Part:BBa K5398005"

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====Protein self-healing====
 
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<p>We obtained protein samples of TRn5 by freezedrying 24 h (Fig. 4).</p>
  
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        <img src="https://static.igem.wiki/teams/5398/trn5/freezedrying.webp" width="500" height="auto" alt="Protein purification">
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        <p><b>Fig. 4 The protein sample freeze-dried by a lyophilizer.</b></p>
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<p>Next, we dissolved protein samples in 5% acetic acid to reach 20 mg/μL, cast into square models and dried them at 70℃ for 3 h to obtain protein films.
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To examine the property of self-healing of TRn5, we punctured a TRn5 protein film to create a hole defect by a needle (Fig. 5a). After putting the punctured film at room temperature for 1 day, we clearly saw the hole defect healing (Fig. 5b).So it was proved that TRn5 has a self-healing property.</p>
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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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    <title>模块示例</title>
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    <style>
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        .module {
 +
*            border: 1px solid #ccc; /
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*            padding: 20px; /
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*            margin: 20px auto; /
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*            width: 500px; /
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*            text-align: center; /
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*            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /
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        }
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        <img src="https://static.igem.wiki/teams/5398/trn5/self-healing-of-trn5-protein-films.webp" width="600" height="auto" alt="Protein purification">
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        <p><b>Fig. 5 Self-healing of TRn5 protein films after puncture damage. .</b></p>
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    <p>a. A hole defect was left by a needle through the film; b. Puncture damage was healed.</p>
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Revision as of 07:50, 19 September 2024


pET29a(+)-TRn5

In order to obtain proteins with self-healing properties, we used the pET29a(+) vector to express TRn5 ( BBa_K5398001) ). 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).

Protein purification

Fig. 1 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 and renatured it, 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. 2).

模块示例

Protein purification

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.

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, thereby confirming the success of our purification approach (Figure 3).

模块示例

Protein purification

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

We obtained protein samples of TRn5 by freezedrying 24 h (Fig. 4).

模块示例

Protein purification

Fig. 4 The protein sample freeze-dried by a lyophilizer.

Next, we dissolved protein samples in 5% acetic acid to reach 20 mg/μL, cast into square models and dried them at 70℃ for 3 h to obtain protein films. To examine the property of self-healing of TRn5, we punctured a TRn5 protein film to create a hole defect by a needle (Fig. 5a). After putting the punctured film at room temperature for 1 day, we clearly saw the hole defect healing (Fig. 5b).So it was proved that TRn5 has a self-healing property.

模块示例

Protein purification

Fig. 5 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.

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]

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.