Difference between revisions of "Part:BBa K5166000"
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     <iframe title="BIT-China: NBP1-1 (2024)" width="560" height="315" src="https://video.igem.org/videos/embed/1a29c57b-e45b-45de-a1ac-3562a1394396?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 NBP1.</p>
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     <iframe title="BIT-China: NBP1-2 (2024)" width="560" height="315" src="https://video.igem.org/videos/embed/f9677562-1250-45e4-8e3e-a4038d41bd9d?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 NBP1 binding 1 Ni<sup>2+</sup>.</p>
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<p style="text-align: center;"><b>Vid. 3</b> Structure of NBP1 binding 3 Ni<sup>2+</sup>s.</p>
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<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 NBP1 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 NBP1 metal-binding peptide gene as the target gene, we obtained the corresponding surface display plasmid.<br>
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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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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>
 
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>
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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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<p style="text-align: center;"><b>Fig. 2</b> Electrophoretic map of engineered yeast after colony PCR.</p>
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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><br>
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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>
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<h1>Reference</h1>
 
<h1>Reference</h1>

Revision as of 08:10, 1 October 2024

Nickel-Binding Peptide 1

Usage

    Nickel-binding peptide 1 (NBP1) is a nickel-binding peptide. It can bind Ni(II) specificaly. We display it with Pichia pastoris through cell-surface display systems.


Biology

    NBP1 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 NBP1: GLHTWATNLYHM. 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 NBP1.


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

Vid. 2 Structure of NBP1 binding 1 Ni2+.


    It can be seen that the atoms coordinating with Ni2+ are mainly O 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, three Ni2+ ions were added to the molecular model, and four Cl- ions were added to balance their charge. The total charge of the system was +2. Through structural optimization, we get the following results:
Video Embed

Vid. 3 Structure of NBP1 binding 3 Ni2+s.


    It can be seen that the counterion Cl- participates in the coordination, and the counterion also binds to the carbon skeleton, which can regulate the charge distribution and the structure of the polypeptide. The two Ni2+ are connected by O-bridges wrapped in a polypeptide, and may form clusters in other situations.


Experiments

1.We used Pichia Pastoris GS115 as chassis cell and pGAPZα plasmid to design the display system. By inserting the NBP1 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, et al. Enhanced Biosorption of Nickel Ions on Immobilized Surface-Engineered Yeast Using Nickel-Binding Peptides[J]. Front Microbiol, 2019,10:1254.
[2]Jmol: an open-source Java viewer for chemical structures in 3D. http://www.jmol.org/
[3] Q. Sun, et al. J. Chem. Phys. 2020, 153, 024109
[4] Q. Sun, et al. WIREs Comput. Mol. Sci. 2018, 8, e1340
[5] Q. Sun, J. Comp. Chem. 2015, 36, 1664
[6] L.-P. Wang, C. C. Song, J. Chem. Phys. 2016, 144, 214108

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]