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

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       <ul><li><a href="https://2018.igem.org/Team:Nanjing-China/Design">Design</a></li></ul></div>
 
       <ul><li><a href="https://2018.igem.org/Team:Nanjing-China/Design">Design</a></li></ul></div>
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    <li><a href="#cds"><font size="-1">Biosynthesis of CdS</font></a></li>
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  <li><a href="# nitrogen"><font size="-1">Nitrogen fixation</font></a></li>
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    <li><a href="#device">Device</a></li>
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       <ul><li><a href="https://2018.igem.org/Team:Nanjing-China/Notebook">Notebook</a></ul></li></div>
 
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       <ul><li><a href="https://2018.igem.org/Team:Nanjing-China/Demonstrate">Demonstrate</a></ul></li></div>
 
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                 <li><a href="https://2018.igem.org/Team:Nanjing-China/Results">Results</a></li>
 
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             <li><a href="https://2018.igem.org/Team:Nanjing-China/Model">Model</a></li>
 
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                 <li><a href="https://2018.igem.org/Team:Nanjing-China/InterLab">InterLab</a></li>
 
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    <p>Liying Wang et. demonstrated a minimal nitrogen  fixation gene cluster from Paenibacillus sp.WLY 78 which was compried of Pnif  promoter and nine structural genes. Inspired by this study, we transferred this  gene cluster to E.coli to create engineered E.coli cells which are capable of  producing active nitrogenase. We achieved this by extracting the gene cluster  from Paenibacillus sp.WLY78, connecting it to plasmid Psb1C3 and transformed it  to E coli cells.In order to ensure the expression of this gene cluster in E  coli,first we verified the transcriptional activity of Pnif promoter in E coli  cells by conducting control experiments.In the test group,we replaced the  native T5 promoter on pQE80L vector with Pnif,connected it to Dronpa fluorescent  protein gene and transformed the new vectors to E.coli cells.In the control  group, pQE80L vectors with T5 promoter and Dronpa gene were transformed to E  coli cells. The comparable level of fluorescence intensity of the two groups  indicated that Pnif promoter is transcriptional active in E coli cells.</p>
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<h2>Biosynthesis of CdS semiconductor</h2>
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<p>To construct our light-driven system, we first induce the precipitation of CdS semiconductor on the cell membrane. Two plasmids encoding the surface display protein OmpA-PbrR and the nitrogenase are co-transferred into <em>E. coli</em> strain. After Cd2+ is added into the media, the ions specifically bind to PbrR leading to aggregation of Cd2+ ions. At last when S2- ions are added into the media, <em>E. coli</em> cells form CdS semiconductor on the cell membrane because of the aggregation.</p></div>
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<p>Simultaneously, in order to achieve in site synthesis of CdS nanocrystals on cell surfaces,we transformed the vector with opmA-PbrR gene to E.coli cells.This gene encodes opmA-PbrR protein complex,which can be fixed on cell surface by outer membrane protein (OMP) opmA.The function of PbrR protein is to adsorb Cd2+ in the environment and further form CdS nanocrystal on cell surface,a key component of this light-harvesting system. When this system is exposed by light, electrons from electron donor conduct transition while CdS nanocrystals on cell surfaces transfer high-energy electrons to Mo-Fe protein subunit of nitrogenase.Mo-Fe protein then  utilizes the energy from these electrons rather than ATP to reduce dinitrogen into ammonia. With the method mentioned above, we successfully constructed a whole-cell light-driven nitrogen fixation system.</p></div>
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<h2>Light-driven nitrogen fixation in E. coli cells</h2>
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<p>To address the problem of electron transduction, CdS semiconductor  act as semiconductors imitating the photosynthetic system under illumination.  It provided excited electrons to <u>a redox mediator methyl viologen (MV) </u>which  then penetrates into <em>E. coli</em> cells  and transfer the electrons to Mo-Fe protein subunit of nitrogenase. Mo-Fe  protein then utilizes the energy from these electrons to reduce dinitrogen to  ammonia. The semiconductor regains its lost electron from sacrificial electron  donors.<br />
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  As a part of biohybrid system, the PbrR protein bears a high  specificity. Our system is supposed to self-repair and can be built with a  rather low cost. This design is of general applications as OmpA protein is only  a surface display machinery for <em>E. coli</em>. <br />
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  This part of the system is the expansion of our hydrogen production,  and it proves that surface display machinery can be expanded to a general  principle for biohybrid photosynthesis.</p></div>
 
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<p>In later period of our study, in order to elevate the electron transfer efficiency, we introduced Ag2S to realize Ag2S CdS cocatalysis. We respectively introduced ompA-spy tag Ag oligopeptide complex and ompA-spychcatcher-PbrR complex to E.coli cells. In that way, two types of E coli cells--with Ag2S or CdS nanocrystals adsorbed on surfaces specifically bind with each other through covalent bonds between spytag and spycatcher. Eventually, nitrogen fixation efficiency of our system showed a remarkable increase</p>
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<p>To apply our system to the real world, we also designed a <a href="https://2018.igem.org/Team:Nanjing-China/Device">device</a> consists of 3 modules: incubation module, illumination module and control module.</p></div>
 
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Revision as of 10:12, 22 September 2018

Nanjing-China2018

Biosynthesis of CdS semiconductor

To construct our light-driven system, we first induce the precipitation of CdS semiconductor on the cell membrane. Two plasmids encoding the surface display protein OmpA-PbrR and the nitrogenase are co-transferred into E. coli strain. After Cd2+ is added into the media, the ions specifically bind to PbrR leading to aggregation of Cd2+ ions. At last when S2- ions are added into the media, E. coli cells form CdS semiconductor on the cell membrane because of the aggregation.

play
stop

Light-driven nitrogen fixation in E. coli cells

To address the problem of electron transduction, CdS semiconductor act as semiconductors imitating the photosynthetic system under illumination. It provided excited electrons to a redox mediator methyl viologen (MV) which then penetrates into E. coli cells and transfer the electrons to Mo-Fe protein subunit of nitrogenase. Mo-Fe protein then utilizes the energy from these electrons to reduce dinitrogen to ammonia. The semiconductor regains its lost electron from sacrificial electron donors.
As a part of biohybrid system, the PbrR protein bears a high specificity. Our system is supposed to self-repair and can be built with a rather low cost. This design is of general applications as OmpA protein is only a surface display machinery for E. coli.
This part of the system is the expansion of our hydrogen production, and it proves that surface display machinery can be expanded to a general principle for biohybrid photosynthesis.

play
stop

Device

To apply our system to the real world, we also designed a device consists of 3 modules: incubation module, illumination module and control module.

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

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