Difference between revisions of "Team:Nanjing-China/Demonstrate"

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       <ul><li><a href="https://2018.igem.org/Team:Nanjing-China/Results">Results</a></ul></li></div>
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       <ul><li><a href="https://2018.igem.org/Team:Nanjing-China/Demonstrate">Demonstrate</a></li></ul></div>
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    <li><a href="#cds">CdS</a></li>
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      <li><a href="#overview">Overview</a></li>
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  <li><a href="#nit"><font size="-1">Nitrogen fixation</font></a></li>
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      <li><a href="#cds">CdS</a></li>
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      <li><a href="#nit"><font size="-1">Nitrogen fixation</font></a></li>
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        <h2>Biosynthesis of CdS semiconductor</h2>
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      <h2>Overview</h2>
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    <img src="https://static.igem.org/mediawiki/2018/a/a7/T--Nanjing-China--TEX-EDX.jpg" width="100%" />
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        <p>This year, we found  an economic, efficient and environmental-friendly way to utilize nitrogen in  the air. Our design consists of three parts: biosynthesis of CdS semiconductor, light-driven  nitrogen fixation and reaction device. All three parts have gone  through in-depth examination and successfully work out under real conditions.<br />
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    <p>TEM-EDX analysis of CdS semiconductor. a) TEM images of biosynthesized CdS semiconductor on the surface of an engineered E. coli cell. b) Elemental analysis using EDX system, the result show that the semiconductor on cell surface is mainly composed of cadmium and sulfide.</p>
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         The following list exhibits our key  achievements in this project.<br />
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    <p>&nbsp;</p>
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        Constructed and  tested engineered <em>E. coli</em> which express nitrogenase and OmpA-PbrR. <br />
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    <img src="https://static.igem.org/mediawiki/2018/e/e1/T--Nanjing-China--toxicity-1.jpg" width="70%" />
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        Constructed and  tested light-driven system based on OmpA-PbrR protein.<br />
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        Achieved  light-driven nitrogen fixation.<br />
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    <img src="https://static.igem.org/mediawiki/2018/3/3e/T--Nanjing-China--toxicity-2.jpg" width="70%" />
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        Designed a reaction  device that cater for our light-driven nitrogen fixation reaction.</p>
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<p>Toxicity test was conducted to determine the maximum amount of Cd<sup>2+</sup> that is agreeable for E. coli growth. Compared with the control group that doesn’t contain surface-display gene, our constructed E. coli strain is more sensitive to Cd<sup>2+</sup>, and its growth will be restricted When the Cd<sup>2+</sup> concentration is above 150μM. So we select 100μM as the final Cd<sup>2+</sup> concentration for our further assays.</p>
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    <p>The amount of biosynthesized CdS semiconductor on the E. coli cell surface was measured using inductively coupled plasma mass spectrometry (ICP-MS). These data confirmed the surface-displayed PbrR-mediated biological precipitation of CdS semiconductor on the outer membranes of the cells.</p>
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    <p>We performed ultraviolet-visible (UV-vis) spectral measurements to directly determine the optical band gap energy of these CdS semiconductor and the photocatalytic capability for the biological precipitation of CdS semiconductor on the outer membranes of the bacterial cells. The lowest-energy transition of the biosynthesized CdS nanoparticles was detected in the visible region of the solar spectrum (Eg = 2.92 eV, labsorption = 424 nm), confirming the photocatalytic ability of the in situ biosynthesized CdS semiconductor.</p>
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    <h2>Light-driven nitrogen fixation in E. coli cells</h2>
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      <h3>Biosynthesis  of CdS semiconductor</h3>
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      <p>One key element  of light-driven system in our design is CdS semiconductor precipitated by the OmpA-PbrR  fused protein. We used TEM-EDX analysis to characterize CdS semiconductor  precipitated on E. coli cell. We also determined the maximum concentration of  Cd2+ appropriate for strain growth, as well as the amount of CdS  that can be precipitated on cell surface.</p>
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<p>To verify the expression of nitrogenase gene, we conducted Real-time Quantitative PCR(QPCR) to detect the transcription level of nif gene cluster in engineered E. coli, using 16S DNA as an internal reference. The result provided the relative expression level of each nif gene in our constructed E. coli strain. After comparing the result with the ideal expression ratio in Paenibacillus CR1 and model the transcription, we plan to optimize the nif gene cluster by adding promoters or altering the position of genes.<br/>
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Nitrogenase can not only reduce dinitrogen to ammonia but also reduce ethylene to acetylene. Therefore, we use gas chromatography to detect the amount of acetylene reduced, and indirectly detect its nitrogen fixation activity. On the basis of these results, NH<sub>3</sub> production by our engineered E. coli cell–CdS hybrid system is directly related to the biosynthesized CdS semiconductors as well as illumination and anaerobic conditions.</p>
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      <h3>Light-driven  nitrogen fixation</h3>
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        <p>CdS  semiconductor generate excited electrons under illumination which are then  passed to nitrogenase for nitrogen fixation. We introduce nitrogenase to <em>E.coli</em> to enable it to reduce dinitrogen to ammonia. We conducted QPCR to detect  relative transcriptional level of each <em>nif</em> gene. We also plan to optimize the structure of <em>nif </em>gene operon after modeling. Using acetylene reduce assay, we then  verify the activity of nitrogenase in our system.</p>
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      <img src="https://static.igem.org/mediawiki/2018/c/cf/T--Nanjing-China--QPCR1.jpg" width="40%" />
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      <h3>Reaction  device</h3>
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        <p>We designed a light-driven microbe reaction  device to apply our system to practical use. After a few test, we proved our  device to be quite practical. <a href="https://2018.igem.org/Team:Nanjing-China/Hardware">(see device for more details)</a></p>
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Revision as of 11:48, 11 October 2018

Nanjing-China2018

Overview

This year, we found an economic, efficient and environmental-friendly way to utilize nitrogen in the air. Our design consists of three parts: biosynthesis of CdS semiconductor, light-driven nitrogen fixation and reaction device. All three parts have gone through in-depth examination and successfully work out under real conditions.
The following list exhibits our key achievements in this project.
Constructed and tested engineered E. coli which express nitrogenase and OmpA-PbrR.
Constructed and tested light-driven system based on OmpA-PbrR protein.
Achieved light-driven nitrogen fixation.
Designed a reaction device that cater for our light-driven nitrogen fixation reaction.

Biosynthesis of CdS semiconductor

One key element of light-driven system in our design is CdS semiconductor precipitated by the OmpA-PbrR fused protein. We used TEM-EDX analysis to characterize CdS semiconductor precipitated on E. coli cell. We also determined the maximum concentration of Cd2+ appropriate for strain growth, as well as the amount of CdS that can be precipitated on cell surface.

Light-driven nitrogen fixation

CdS semiconductor generate excited electrons under illumination which are then passed to nitrogenase for nitrogen fixation. We introduce nitrogenase to E.coli to enable it to reduce dinitrogen to ammonia. We conducted QPCR to detect relative transcriptional level of each nif gene. We also plan to optimize the structure of nif gene operon after modeling. Using acetylene reduce assay, we then verify the activity of nitrogenase in our system.

Reaction device

We designed a light-driven microbe reaction device to apply our system to practical use. After a few test, we proved our device to be quite practical. (see device for more details)

Address

Life Science Department
#163 Xianlin Blvd, Qixia District
Nanjing University
Nanjing, Jiangsu Province
P.R. of China
Zip: 210046

Email

nanjing_china@163.com

Social media

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iGEM Nanjing-China@nanjing_china