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

We first verified the activity of the nitrogen-fixing gene cluster promoter Pnif in E. coli. The expression of surface display proteins and nitrogenases in E. coli was then determined by SDS electrophoresis. Finally, the nitrogenase activity was detected by acetylene reduction assay, colorimetric assay and fluorescence assay using o-dialdehyde.

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.

Transfer PQE80L with Pnif and Dronpa into E.coli BL21. Pick up the plasmid from the contracted E.coli, dual-enzyme digest and make the DNA elestrophoresis, All the cannel are from the contracted E.coli. Each one has two sequence and the shorter one is about 700bp, which proves that the plasmid with Pnif and dro has been transferred to the E.coli.

Figure1. the DNA electrophoresis of Pnif+dro from contracted E.coli. All the cannel are from the contracted E.coli

Fluorescence and light absorption measurements reflect the change in Pnif promoter activity over time. The red line in the figure represents the change in fluorescence intensity in the presence of the Pnif promoter, while the black line represents the change in fluorescence intensity at the T5 promoter of the plasmid. The activity of the Pnif promoter reached a maximum around 11 hours and remained essentially unchanged thereafter. This result provides a reference incubation time for our further assays.

Figure2. The time curve of nifB promoter activity. The red line in the figure represents the change in fluorescence intensity in the presence of the Pnif promoter, while the black line represents the change in fluorescence intensity at the T5 promoter of the plasmid. The activity of the Pnif promoter reached a maximum around 11 hours and remained essentially unchanged thereafter. This result provides a reference incubation time for our further assays.

Using a similar method, we detected the activity of the Pnif at different ammonium concentrations. The results show that Pnif is not sensitive to ammonium.

Fiture3. Activity changes of Pnif promoter at different ammonium concentrations.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine whether the two groups of genes were transferred and expressed specific proteins. SDS-PAGE can separate proteins into several bands according to the different migration rates produced by different molecular masses. Three lanes on the left are proteins from E.coli cultured in non-nitrogen-fixation condition with O2. While three lanes on the right are proteins from E.coli cultured in nitrogen-fixation condition without O2. On the last lane, we can see the band of OmpA-PbrR, which is approximately 44kDa, beside the first arrow and the band hesA, which is a 24kDa nitrogenase protein, beside the second arrow.

Figure4. SDS of the protein. The protein with “1” is from the E.coli cultured in no nitrogen-fixed condition. The protein with “2” is from the E.coli cultured in nitrogen-fixed condition.On the last lane, we can see the band of OmpA-PbrR, which is approximately 44kDa, beside the first arrow and the band hesA, which is a 24kDa nitrogenase protein, beside the second arrow.

Nitrogenase can only reduce nitrogen 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. P and N represent E. coli containing the surface display gene and the nitrogen fixation gene, respectively, and the two groups labeled with AR are grown under oxygen-free nitrogen fixation conditions. Through the experimental results, we can conclude that under nitrogen fixation conditions, our constructed E. coli can exhibit a certain nitrogenase activity.

Figure5. The reduction of acetylene. P and N represent E. coli containing the surface display gene and the nitrogen fixation gene, respectively, and the two groups labeled with AR are grown under oxygen-free nitrogen fixation conditions.

The amount of NH₄⁺ produced by the nitrogenase was measured using a colorimetric assay kit. After the reaction under assay conditions, the absorption peak at 570 nm is proportional to the amount of NH4+ in the reaction solution.
First, we use the NH4Cl standard solution provided by the kit to make a standard curve. As shown in Figure a, the relationship between the absorption intensity of OD570 and the NH4+ concentration is calculated according to the formula obtained from the standard curve.
Figure b is the amount of NH₄⁺ produced by E. coli under different culture conditions after conversion according to the standard curve. Four groups of E. coli were determined under nitrogen-free nitrogen fixation conditions. Approximately 2.71 nmol of NH₄⁺ is produced per 1 million E. coli

Figure6a.standard curve of the Ammonium Chloride standard NH4Cl solution

Figure6b. the OD570 and OD600 of the ompa-pbrr, nif and ompa-pbrr+nif(light/dark) in an oxygen-free environment

In Figure a, the relationship between the absorption intensity of OD570 and the NH4+ concentration is calculated according to the formula obtained from the standard curve. Figure b is the amount of NH4+ produced by E. coli under different culture conditions after conversion according to the standard curve. Four groups of E. coli were determined under nitrogen-free nitrogen fixation conditions. Approximately 2.71 nmol of NH4+ is produced per 1 million E. coli