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

To demonstrate that our design, we constructed E. coli JM109 strain containing both nif gene cluster and OmpA-PbrR gene (abbreviated as EJNC in the following passage) as the experimental group. We also set E. coli JM109 (abbreviated as EJ) that doesn’t contain none of the genes as a control group.

Biosynthesis of CdS semiconductor on cell surface

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

In order to determine the optimum ²⁺ concentration of the system, we conducted a ²⁺ toxicity test. The existence of cadmium ions shows no significant toxic effects on both strains which is evidenced by the virtually equal colony forming units, though a short term growth readjustment brought by heterologous gene expression does occur in EJNC. Considering that, we select 150 μM as the ²⁺ concentration for our follow-up experiments.

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

The amount of biosynthesized CdS semiconductor on the E. coli cell surface was measured using inductively coupled plasma mass spectrometry (ICP-MS). It confirms the surface-displayed PbrR-mediated biological precipitation of CdS semiconductor on the outer membranes of cells.

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.

To acquire a fuller understanding of CdS nanoparticles’ characteristics, we performed Transmission electron microscopy analyze of CdS nanoparticles. The morphology and particle size of CdS nanoparticles are shown in TEM images. Next, we conducted energy-dispersive x-ray spectroscopy (EDX) to analyze its elemental composition. The result demonstrates that the semiconductor on cell surface is mainly composed of cadmium and sulfide.

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.

We performed ultraviolet-visible (UV-vis) spectral measurements to directly determine the optical band gap and photocatalytic capability of these CdS semiconductor. The lowest-energy transition of the biosynthesized CdS nanoparticles was detected in the visible region of the solar spectrum (Eg = 2.92 eV, λ_absorption = 424 nm), verifying its photocatalytic ability.

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⁺. 

The redox dye methylviologen (MV²⁺) is a well-established electron mediator. In combination with MV²⁺, the E. coli/CdS hybrids system easily serve as a biocatalyst for photosynthesis. The concentrations of reduced MV in various experimental groups were measured under anaerobic conditions, confirming that CdS semiconductor transfers an electron to MV²⁺ for every photon it absorbs.

Light-driven nitrogen fixation in E. coli cells

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.

To verify the expression of nif gene cluster, we conducted Real-time Quantitative PCR(qRT-PCR) to detect the transcriptional level of each nif gene in engineered E. coli, using 16S DNA as an internal reference. The result provide the relative expression level of each nif gene in our constructed E. coli strain. After comparing the result with the nif gene cluster expression in Paenibacillus polymyxa CR1, we modeled the transcription and planned to optimize the structure of nif gene cluster to achieve the best stoichiometric proportion of each nif gene. (see modeling for more details)

Nitrogenase can not only reduce dinitrogen to ammonia, but also reduce ethylene to acetylene. Therefore, we performed acetylene reduce to detect the amount of acetylene reduced, and indirectly detected its nitrogen fixation activity. On the basis of these results, NH3 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.