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

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 on cell surface

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.

Figure 1. Cd²⁺ toxicity test. Cadmium ions shows no significant toxic effects on both strains.

Figure 2. Cd²⁺ absorption test. The introduction of OmpA-PbrR confers the host cell with Cd²⁺ absorption capacity.

Figure 3. (a) TEM images of biosynthesized CdS nanoparticles on the surface of a EJNC cell. (b) EDX confirmation of randomly chosen CdS nanoparticle. The absorbed Cd²⁺ precipitates on the outer membrane of EJNC in the form of CdS nanoparticles.

Figure 4. Characterization of biologically precipitated CdS nanoparticles on the outer membranes of E. coli cells. The UV-Vis Spectrum of E. coli/CdS hybrids in solution demonstrating a band gap at 424 nm.

Figure 5. Quantitative comparison of the photoelectrical capacity of in situ biosynthesized CdS nanoparticles. The concentrations of reduced methylviologen (MV) in various experimental groups confirm that the CdS nanoparticles precipitate on the EJNC cells adsorb a photon and transfer an electron to MV²⁺. 

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.

Figure 6. Expression profiles of each structure gene in the nif cluster that overexpressed in EJNC. Relative expression compared to the housekeeping gene 16S rRNA is shown. qRT-PCR analysis demonstrates that all the component genes of the nif cluster are significantly over expressed in EJNC whereas the transcription of these genes are no detected (N.D.) in nondiazotrophic E. coli JM109.

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)