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>
 
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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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      <p>Our design is composed of three parts: biosynthesis of CdS semiconductor, light-driven nitrogen fixation and a light-driven biohybrid reaction device. This system is the expansion of our previous project of hydrogen production (Nanjing-China 2016), and it proves that surface display mechanism is capable of being expanded to a general principle for light-driven biohybrid reactions.</p>
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<p align="left">To construct our light-driven system, we induce the in situ synthesis of CdS semiconductor on the <em>E. coli</em> cell surface. One key element of our system is fused protein OmpA-PbrR. OmpA (Outer membrane  protein A) fixes the protein complex on outer cell membrane while PbrR (lead-specific binding protein) adsorbs Cd<sup>2+</sup> in the environment and further forms CdS  semiconductor on cell surface.</p>
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<p> After Cd<sup>2+</sup> ions are added  into the culture, the ions specifically bind to PbrR protein contributing to the  aggregation of Cd<sup>2+</sup>. Combining with S<sup>2-</sup> ions in the  media, CdS semiconductors are therefore formed on the outer membrane of cells.</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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<p>When the system is exposed in light, electrons of  the CdS semiconductor conduct  transit, and CdS provide these  excited electrons to the electrons to Mo-Fe protein subunit of nitrogenase. Subsequently, the Mo-Fe protein utilizes the energy from these  electrons to reduce N<sub>2</sub>(dinitrogen) to NH<sub>3</sub>(ammonia). Finally, the semiconductor regains its lost electron from sacrificial electron donors.</p>
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<p>This design is of general applications as OmpA  protein is merely a surface display mechanism for&nbsp;<em>E. coli</em>, and PbrR can be  replaced with other proteins with different specificity. </p>
 
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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>We also designed a light-driven biohybrid 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 hardware for more details)</a> </p></div>
 
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Latest revision as of 03:02, 18 October 2018

Nanjing-China2018

Overview

Our design is composed of three parts: biosynthesis of CdS semiconductor, light-driven nitrogen fixation and a light-driven biohybrid reaction device. This system is the expansion of our previous project of hydrogen production (Nanjing-China 2016), and it proves that surface display mechanism is capable of being expanded to a general principle for light-driven biohybrid reactions.

Biosynthesis of CdS semiconductor on cell surface

To construct our light-driven system, we induce the in situ synthesis of CdS semiconductor on the E. coli cell surface. One key element of our system is fused protein OmpA-PbrR. OmpA (Outer membrane protein A) fixes the protein complex on outer cell membrane while PbrR (lead-specific binding protein) adsorbs Cd2+ in the environment and further forms CdS semiconductor on cell surface.

After Cd2+ ions are added into the culture, the ions specifically bind to PbrR protein contributing to the aggregation of Cd2+. Combining with S2- ions in the media, CdS semiconductors are therefore formed on the outer membrane of cells.

play
stop

Light-driven nitrogen fixation in E. coli cells

When the system is exposed in light, electrons of the CdS semiconductor conduct transit, and CdS provide these excited electrons to the electrons to Mo-Fe protein subunit of nitrogenase. Subsequently, the Mo-Fe protein utilizes the energy from these electrons to reduce N2(dinitrogen) to NH3(ammonia). Finally, the semiconductor regains its lost electron from sacrificial electron donors.

This design is of general applications as OmpA protein is merely a surface display mechanism for E. coli, and PbrR can be replaced with other proteins with different specificity.

play
stop

Reaction device

We also designed a light-driven biohybrid reaction device to apply our system to practical use. After a few test, we proved our device to be quite practical. (see hardware 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