Difference between revisions of "Part:BBa K5166001"
 
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<h1>Usage</h1>
 
<h1>Usage</h1>
  
−
<p>&nbsp;&nbsp;&nbsp;&nbsp;Nickel-binding peptide 2 (NBP2) is a nickel-binding peptide. It can bind Ni(II) specificaly. We display it with <i>Pichia pastoris</i> through cell-surface display systems.
+
<p>Nickel-binding peptide 2 (NBP2) is a nickel-binding peptide. It can bind Ni(II) specificaly. We display it with <i>Pichia pastoris</i> through cell-surface display systems.
 
</p>
 
</p>
  
  
 
<h1>Biology</h1>
 
<h1>Biology</h1>
−
<p>&nbsp;&nbsp;&nbsp;&nbsp;NBP2 is selected from Phage Display Library Screening: The Ph.D.-12 peptide library kit (E8110S, New England Bio Labs, USA). A study has shown that the sorption of nickel ions on the surface of yeast cells increased with the increasing number of nickel Ni(II)-binding peptides displayed. The combined expression of the three peptides(NBP1+NBP2+NBP3) by <i>EBY100/pYD1-N123</i> demonstrated the highest sorption of Ni(II) (2.603 ± 0.004 g g−1, dry weight) and an enhanced sorption capacity of 60.15%, compared to <i>S. cerevisiae EBY100</i>[1].</p>
+
<p>NBP2 is selected from Phage Display Library Screening: The Ph.D.-12 peptide library kit (E8110S, New England Bio Labs, USA). A study has shown that the sorption of nickel ions on the surface of yeast cells increased with the increasing number of nickel Ni(II)-binding peptides displayed. The combined expression of the three peptides(NBP1+NBP2+NBP3) by <i>EBY100/pYD1-N123</i> demonstrated the highest sorption of Ni(II) (2.603 ± 0.004 g g−1, dry weight) and an enhanced sorption capacity of 60.15%, compared to <i>S. cerevisiae EBY100</i><sup>[1]</sup>.</p>
  
  
 
<h1>Simulation</h1>
 
<h1>Simulation</h1>
−
<p> &nbsp;&nbsp;&nbsp;&nbsp;We used the MLatom calculation program on the XACS platform to perform structural calculations of the binding between metal ion binding peptides and metal ions[2-6], in order to predict the binding ability.  
+
<p> We used the MLatom calculation program on the XACS platform to perform structural calculations of the binding between metal ion binding peptides and metal ions<sup>[2-6]</sup>, in order to predict the binding ability.  
 
In the following video: white is H, gray is C, blue is N, red is O, flesh color is Co, purple is Li, light green is Cl, dark green is Ni.<br>
 
In the following video: white is H, gray is C, blue is N, red is O, flesh color is Co, purple is Li, light green is Cl, dark green is Ni.<br>
 
(1) Sequence<br>
 
(1) Sequence<br>
−
&nbsp;&nbsp;&nbsp;&nbsp;Here we perform machine learning quantum chemistry calculations for the sequence NBP2: HAVSPTLPAYSK. Firstly, the geometric structure of the polypeptide chain was optimized, and the folded configuration was obtained, as shown below.<br>
+
Here we perform machine learning quantum chemistry calculations for the sequence NBP2: HAVSPTLPAYSK. Firstly, the geometric structure of the polypeptide chain was optimized, and the folded configuration was obtained, as shown below.<br>
 
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 +
  <iframe title="BIT-China: NBP2 (2024)" width="560" height="315" src="https://video.igem.org/videos/embed/fd62692c-bef0-4d19-baef-56ba06962247?loop=1&amp;autoplay=1&amp;title=0&amp;warningTitle=0&amp;peertubeLink=0&amp;p2p=0" frameborder="0" allowfullscreen="" sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
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<p style="text-align: center;"><b>Vid. 1</b> Structure of NBP2.</p >
 +
 
</body>
 
</body>
 
</html><br>
 
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(2) Structural analysis of single ion binding<br>
 
(2) Structural analysis of single ion binding<br>
−
&nbsp;&nbsp;&nbsp;&nbsp;We simulated the binding of a single Ni ion to a folded polypeptide chain. Through structural optimization, we get the following results:<br><br>
+
We simulated the binding of a single Ni ion to a folded polypeptide chain. Through structural optimization, we get the following results:<br>
 
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   <div class="iframe-container">
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 +
  <iframe title="BIT-China: NBP2Ni1 (2024)" width="560" height="315" src="https://video.igem.org/videos/embed/de4f007d-5ef8-4acf-acc6-38642e081b7a?loop=1&amp;autoplay=1&amp;title=0&amp;warningTitle=0&amp;peertubeLink=0&amp;p2p=0" frameborder="0" allowfullscreen="" sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
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<p style="text-align: center;"><b>Vid. 2</b> Structure of NBP2 binding 1 Ni<sup>2+</sup>.</p >
 +
 
</body>
 
</body>
 
</html><br>
 
</html><br>
−
&nbsp;&nbsp;&nbsp;&nbsp;It can be seen that the atoms coordinating with Ni2+ are mainly O and N atoms on the polypeptide chain. The Ni2+ ion is encased.<br>
+
It can be seen that the atoms coordinating with Ni<sup>2+</sup> are mainly O and N atoms on the polypeptide chain. The Ni<sup>2+</sup> ion is encased.<br>
 
(3) The combination of multiple ions<br>
 
(3) The combination of multiple ions<br>
−
&nbsp;&nbsp;&nbsp;&nbsp;We designed this metal-binding peptide in the hope that they could trap multiple metal ions and increase efficiency. In order to analyze the binding of multiple Ni2+ ions, four Ni2+ ions were added to the molecular model, and four Cl- ions were added to balance their charge, with a total charge of +4. Through structural optimization, we get the following results:<br>
+
We designed this metal-binding peptide in the hope that they could trap multiple metal ions and increase efficiency. In order to analyze the binding of multiple Ni<sup>2+</sup> ions, four Ni<sup>2+</sup> ions were added to the molecular model, and four Cl<sup>-</sup> ions were added to balance their charge, with a total charge of +4. Through structural optimization, we get the following results:<br>
 
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    height: 50vh; /* 使整个视口成为一个flex容器 */
 +
    margin: 0; /* 移除默认的边距 */
 +
  }
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  .iframe-container {
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    flex: 1; /* 使容器能够扩展以适应iframe */
 
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     justify-content: center;
 
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+
   <div class="iframe-container">
 +
 
 +
  <iframe title="BIT-China: NBP2Ni4 (2024)" width="560" height="315" src="https://video.igem.org/videos/embed/cb5a0d24-6e93-4f6c-9e1c-2d6dcf6f89d7?loop=1&amp;autoplay=1&amp;title=0&amp;warningTitle=0&amp;peertubeLink=0&amp;p2p=0" frameborder="0" allowfullscreen="" sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
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<p style="text-align: center;"><b>Vid. 3</b> Structure of NBP2 binding 4 Ni<sup>2+</sup>.</p >
 +
 
</body>
 
</body>
 
</html><br>
 
</html><br>
−
&nbsp;&nbsp;&nbsp;&nbsp;It can be seen that the N and carbonyl O atoms of higher amines participate in the coordination, the counterion Cl- participates in the coordination, and the counterion also interacts with the carbon skeleton, which can regulate the overall charge distribution of the polypeptide chain, regulate the structure of the polypeptide, and stabilize the system.</p>
+
It can be seen that the N and carbonyl O atoms of higher amines participate in the coordination, the counterion Cl<sup>-</sup> participates in the coordination, and the counterion also interacts with the carbon skeleton, which can regulate the overall charge distribution of the polypeptide chain, regulate the structure of the polypeptide, and stabilize the system.</p>
  
  
 
<h1>Experiments</h1>
 
<h1>Experiments</h1>
−
<p>1.We used <i>Pichia Pastoris</i> GS115 as chassis cell and pGAPZα plasmid to design the display system. By inserting the NBP2 metal-binding peptide gene as the target gene, we obtained the corresponding surface display plasmid.<br>
+
<p>1.We used <i>Pichia Pastoris</i> GS115 as chassis cell and pGAPZα plasmid to design the display system. By inserting the NBP2 metal-binding peptide gene as the target gene, we obtained the corresponding surface display plasmid.<br><br>
−
2.After constructing the plasmid, we introduced it into <i>Escherichia coli</i> for amplification. After amplification, the plasmids were extracted and purified, and sent for sequencing. After obtaining the correct sequencing results, the plasmid was transformed into <i>Pichia pastoris</i> by means of electrical stimulation. Finally, by colony PCR, we determined that the plasmid was successfully introduced into the yeast.<br>
+
<html>
−
3.After obtaining the engineered yeast, we designed some experimental schemes to qualitatively test their adsorption effect on target metal ions. In order to test the adsorption effect, the engineered yeast and the prepared single metal solution were mixed according to a certain proportion, removed and centrifuged after 2 hours, and the supernatant and precipitation were stored respectively. We chose to use a graphite furnace to detect the concentration of metal ions in the supernatant. By calculating the ratio of the reduced metal concentration in the supernatant to the original added metal concentration, we obtained the adsorption rate of the engineered strain on the target metal ions, and made a comparison to select the strain with better adsorption effect.</p>
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  .center img {
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  }
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<div class="center">
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  <img src="https://static.igem.wiki/teams/5166/images/adsorption-group/e-coli-strips.jpg">
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<p style="text-align: center;"><b>Fig. 1</b> Electrophoretic map of plasmids containing MBPs gene.</p >
 +
 
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</body>
 +
</html><br>
 +
2.After constructing the plasmid, we introduced it into <i>Escherichia coli</i> for amplification. After amplification, the plasmids were extracted and purified, and sent for sequencing. After obtaining the correct sequencing results, the plasmid was transformed into <i>Pichia pastoris</i> by means of electrical stimulation. Finally, by colony PCR, we determined that the plasmid was successfully introduced into the yeast.<br><br>
 +
<html>
 +
<head>
 +
<style>
 +
  .center {
 +
    text-align: center;
 +
    display: block; /* 使div变为块级元素 */
 +
    max-width: 100%; /* 设置最大宽度为100%，即容器宽度 */
 +
    height: auto; /* 高度自适应 */
 +
  }
 +
  .center img {
 +
    max-width: 80%; /* 设置图片最大宽度为100%，即容器宽度 */
 +
    height: auto; /* 高度自适应 */
 +
  }
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</style>
 +
</head>
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<body>
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<div class="center">
 +
  <img src="https://static.igem.wiki/teams/5166/images/adsorption-group/yeast-strips.jpg">
 +
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 +
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<p style="text-align: center;"><b>Fig. 2</b> Electrophoretic map of engineered yeast after colony PCR.</p >
 +
 
 +
</body>
 +
</html><br>
 +
3.After obtaining the engineered yeast, we designed some experimental schemes to qualitatively test their adsorption effect on target metal ions. In order to test the adsorption effect, the engineered yeast and the prepared single metal solution were mixed according to a certain proportion, removed and centrifuged after 2 hours, and the supernatant and precipitation were stored respectively. We chose to use a graphite furnace to detect the concentration of metal ions in the supernatant. By calculating the ratio of the reduced metal concentration in the supernatant to the original added metal concentration, we obtained the adsorption rate of the engineered strain on the target metal ions, and made a comparison to select the strain with better adsorption effect.</p><br><html>
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  .unique-container {
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    height: 50vh; /* 根据需要调整容器高度 */
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  }
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  .unique-container img {
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    max-width: 60%; /* 设置图片最大宽度为60%，即原来大小的0.6倍 */
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<body>
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 +
<p style="text-align: center;"><b>Fig. 3</b> The adsorption rate of NBP1-pir，NBP2-pir and NBP4-pir for Ni<sup>2+</sup> within 2 hours.</p >
 +
 
 +
</body>
 +
</html>
  
 
<h1>Reference</h1>
 
<h1>Reference</h1>
−
<p>[1]Li H, Dong W, Liu Y, et al. Enhanced Biosorption of Nickel Ions on Immobilized Surface-Engineered Yeast Using Nickel-Binding Peptides[J]. Front Microbiol, 2019,10:1254.<br>
+
<p>[1]Li, H., Dong, W., Liu, Y., Zhang, H., & Wang, G. (2019). Enhanced Biosorption of Nickel Ions on Immobilized Surface-Engineered Yeast Using Nickel-Binding Peptides. [Journal Article]. Front Microbiol, 10, 1254. doi: 10.3389/fmicb.2019.01254<br>
−
[2]Jmol: an open-source Java viewer for chemical structures in 3D. http://www.jmol.org/<br>
+
[2]Sun, Q., Zhang, X., Banerjee, S., Bao, P., Barbry, M., Blunt, N.,... Chan, G. (2020). Recent developments in the PySCF program package. JOURNAL OF CHEMICAL PHYSICS, 153(2). doi: 10.1063/5.0006074<br>
−
[3] Q. Sun, et al. J. Chem. Phys. 2020, 153, 024109<br>
+
[3] Sun, Q., Berkelbach, T., Blunt, N., Booth, G., Guo, S., Li, Z.,... Chan, G. (2018). PYSCF: the Python-based simulations of chemistry framework. WILEY INTERDISCIPLINARY REVIEWS-COMPUTATIONAL MOLECULAR SCIENCE, 8(1). doi: 10.1002/wcms.1340<br>
−
[4] Q. Sun, et al. WIREs Comput. Mol. Sci. 2018, 8, e1340<br>
+
[4] Sun, Q. (2015). Libcint: An efficient general integral library for Gaussian basis functions. JOURNAL OF COMPUTATIONAL CHEMISTRY, 36(22), 1664-1671. doi: 10.1002/jcc.23981<br>
−
[5] Q. Sun, J. Comp. Chem. 2015, 36, 1664<br>
+
[5] Wang, L., & Song, C. (2022). Geometry optimization made simple with explicit translation and rotation coordinates (vol 144, 214108, 2016). JOURNAL OF CHEMICAL PHYSICS, 157(1). doi: 10.1063/5.0102029<br>
−
[6] L.-P. Wang, C. C. Song, J. Chem. Phys. 2016, 144, 214108</p>
+
[6]Jmol: an open-source Java viewer for chemical structures in 3D. http://www.jmol.org/</p>
  
 
<h1>Sequence and Features</h1>
 
<h1>Sequence and Features</h1>

Latest revision as of 21:04, 1 October 2024

Nickel-Binding Peptide 2

Usage

Nickel-binding peptide 2 (NBP2) is a nickel-binding peptide. It can bind Ni(II) specificaly. We display it with Pichia pastoris through cell-surface display systems.


Biology

NBP2 is selected from Phage Display Library Screening: The Ph.D.-12 peptide library kit (E8110S, New England Bio Labs, USA). A study has shown that the sorption of nickel ions on the surface of yeast cells increased with the increasing number of nickel Ni(II)-binding peptides displayed. The combined expression of the three peptides(NBP1+NBP2+NBP3) by EBY100/pYD1-N123 demonstrated the highest sorption of Ni(II) (2.603 ± 0.004 g g−1, dry weight) and an enhanced sorption capacity of 60.15%, compared to S. cerevisiae EBY100[1].


Simulation

We used the MLatom calculation program on the XACS platform to perform structural calculations of the binding between metal ion binding peptides and metal ions[2-6], in order to predict the binding ability. In the following video: white is H, gray is C, blue is N, red is O, flesh color is Co, purple is Li, light green is Cl, dark green is Ni.
(1) Sequence
Here we perform machine learning quantum chemistry calculations for the sequence NBP2: HAVSPTLPAYSK. Firstly, the geometric structure of the polypeptide chain was optimized, and the folded configuration was obtained, as shown below.
Video Embed

Vid. 1 Structure of NBP2.


(2) Structural analysis of single ion binding
We simulated the binding of a single Ni ion to a folded polypeptide chain. Through structural optimization, we get the following results:
Video Embed

Vid. 2 Structure of NBP2 binding 1 Ni2+.


It can be seen that the atoms coordinating with Ni2+ are mainly O and N atoms on the polypeptide chain. The Ni2+ ion is encased.
(3) The combination of multiple ions
We designed this metal-binding peptide in the hope that they could trap multiple metal ions and increase efficiency. In order to analyze the binding of multiple Ni2+ ions, four Ni2+ ions were added to the molecular model, and four Cl- ions were added to balance their charge, with a total charge of +4. Through structural optimization, we get the following results:
Video Embed

Vid. 3 Structure of NBP2 binding 4 Ni2+.


It can be seen that the N and carbonyl O atoms of higher amines participate in the coordination, the counterion Cl- participates in the coordination, and the counterion also interacts with the carbon skeleton, which can regulate the overall charge distribution of the polypeptide chain, regulate the structure of the polypeptide, and stabilize the system.


Experiments

1.We used Pichia Pastoris GS115 as chassis cell and pGAPZα plasmid to design the display system. By inserting the NBP2 metal-binding peptide gene as the target gene, we obtained the corresponding surface display plasmid.


Fig. 1 Electrophoretic map of plasmids containing MBPs gene.


2.After constructing the plasmid, we introduced it into Escherichia coli for amplification. After amplification, the plasmids were extracted and purified, and sent for sequencing. After obtaining the correct sequencing results, the plasmid was transformed into Pichia pastoris by means of electrical stimulation. Finally, by colony PCR, we determined that the plasmid was successfully introduced into the yeast.


Fig. 2 Electrophoretic map of engineered yeast after colony PCR.


3.After obtaining the engineered yeast, we designed some experimental schemes to qualitatively test their adsorption effect on target metal ions. In order to test the adsorption effect, the engineered yeast and the prepared single metal solution were mixed according to a certain proportion, removed and centrifuged after 2 hours, and the supernatant and precipitation were stored respectively. We chose to use a graphite furnace to detect the concentration of metal ions in the supernatant. By calculating the ratio of the reduced metal concentration in the supernatant to the original added metal concentration, we obtained the adsorption rate of the engineered strain on the target metal ions, and made a comparison to select the strain with better adsorption effect.




Fig. 3 The adsorption rate of NBP1-pir，NBP2-pir and NBP4-pir for Ni2+ within 2 hours.

Reference

[1]Li, H., Dong, W., Liu, Y., Zhang, H., & Wang, G. (2019). Enhanced Biosorption of Nickel Ions on Immobilized Surface-Engineered Yeast Using Nickel-Binding Peptides. [Journal Article]. Front Microbiol, 10, 1254. doi: 10.3389/fmicb.2019.01254
[2]Sun, Q., Zhang, X., Banerjee, S., Bao, P., Barbry, M., Blunt, N.,... Chan, G. (2020). Recent developments in the PySCF program package. JOURNAL OF CHEMICAL PHYSICS, 153(2). doi: 10.1063/5.0006074
[3] Sun, Q., Berkelbach, T., Blunt, N., Booth, G., Guo, S., Li, Z.,... Chan, G. (2018). PYSCF: the Python-based simulations of chemistry framework. WILEY INTERDISCIPLINARY REVIEWS-COMPUTATIONAL MOLECULAR SCIENCE, 8(1). doi: 10.1002/wcms.1340
[4] Sun, Q. (2015). Libcint: An efficient general integral library for Gaussian basis functions. JOURNAL OF COMPUTATIONAL CHEMISTRY, 36(22), 1664-1671. doi: 10.1002/jcc.23981
[5] Wang, L., & Song, C. (2022). Geometry optimization made simple with explicit translation and rotation coordinates (vol 144, 214108, 2016). JOURNAL OF CHEMICAL PHYSICS, 157(1). doi: 10.1063/5.0102029
[6]Jmol: an open-source Java viewer for chemical structures in 3D. http://www.jmol.org/

Sequence and Features


Assembly Compatibility:
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    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]