Team:HBUT-China/Results

Result


Overview of the result

1. Construction of six engineering S. cerevisiae and test function

2. Improvement of nikABCDE

3. Characterization of two EYFP proteins

Test the function of engineering S. cerevisiae

Adsorption effect on simulated Ni2+ concentration (15 mg/L)

In our Human Practical activities, we went to the automobile factory and obtained the nickel ions polluted water samples from the real life. We measured that the nickel ion concentration of the direct effluent discharge was about 15 mg/L. In order to be closer to the real-life situation, we also designed the nickel ion solution with a concentration of 15 mg/L and a suitable yeast dosage of 2 g/L for simulation adsorption experiments.

We carried out adsorption experiments with the original yeast and the six kinds of engineering yeast we constructed at the same time and made sure that the other conditions were exactly the same. We also constructed the original yeast and six engineered yeasts and ensured that the other conditions were identical.

The experimental results are as follows：

Fig 1. Adsorption capacity of simulated Ni2+ (15 mg/L) by engineering yeast after 45 minutes treatment.

Fig 2. Adsorption curve of simulated Ni2+ (15 mg/L) by engineering yeast with time.

We can see that the adsorption capacity of nickel ion in our engineering yeast is higher than that in the original yeast. And the adsorption capacity of S.cerevisiae/BBa_k3126022 (nixA+TgMTP1t2) and S. cerevisiae/BBa_k3126023 (hexa-His+nixA+TgMTP1t2) for nickel ion is stronger, and the removal rate is 80%.

And the adsorption equilibrium qe and adsorption equilibrium constant K were obtained by fitting the enrichment rate model： t/qt=1/(k*qe2)+t/qe


qe(mg/g) Adsorption equilibrium constant K
Original S.cerevisiae 2.44 0.3897
S.cerevisiae/BBa_k3126018(hexa-His) 3.663 0.1696
S.cerevisiae/BBa_k3126019(nixA) 3.3245 0.2817
S.cerevisiae/BBa_k3126020(TgMTP1t2) 3.201 0.4694
S.cerevisiae/BBa_k3126021(hexa-His+nixA) 3.701 0.298
S.cerevisiae/BBa_k3126022(nixA+TgMTP1t2） 5.136 0.135
S.cerevisiae/BBa_k3126023(hexa-His+nixA+TgMTP1t2) 6.34 0.17

From the chart above, we can see that the equilibrium enrichment of engineering yeast has been greatly improved, and the original yeast is easy to reach adsorption saturation.

Adsorption effect on high concentration of Ni2+(200 mg/L)

Through the tolerance experiment of S. cerevisiae to nickel ion, we know that our chassis organism has a strong tolerance to nickel ions, so we increase the concentration of nickel ion solution in the experiment, and further explore the adsorption capacity of our engineering yeast for nickel ion. We increased the concentration of nickel ion solution to 200 mg / L, and the designed yeast dosage was changed to 7g/L.

The experimental results are as follows：

Fig 3. Adsorption capacity of Ni2+ (200 mg/L) by engineering yeast after 45 minutes treatment.


Fig 4. Adsorption curve of simulated Ni2+ (200 mg/L) by engineering yeast with time.

Based on our results, all types of engineering yeasts showed their advantages, and adsorbed more nickel ions compared with original control yeast under the same conditions. Among all the types of engineering yeast, S.cerevisiae/BBa_k3126023 (hexa-His+nixA+TgMTP1t2) have highest absorption effect on nickel ions.

Adsorption effect on actual nickel-containing effluent

The engineering yeast is embedded to absorbed 200 ml, nickel-containing effluent with a concentration of about 15mg/L in the device.

The engineering yeast used is S. cerevisiae/BBa_k3126023 (hexa-His+nixA+TgMTP1t2)

Yeast dosaget: 0.5 g/L×2

Operating process: Place yeast in the treatment pool, and the nickel-containing solution reacted from the injection pool to the treatment pool. After adsorption for 20 min, measure the water samples in the detection pool and take out the immobilization spheres which reached the adsorption equilibrium. Release the new immobilization spheres, After 15 minutes of adsorption, measure the water samples in the detection pool, and finally the waste water is discharged into a water outlet detection pool.

Results: It has been found that yeast can quickly absorb a lot of nickel ions. The nickel concentration in the first batch of water samples after adsorption for 20 ~ 30 minutes was 3.7561 mg/L, and the nickel concentration in the second batch was 0.4659mg/L after 15 minutes of adsorption.

Adsorption of nickel-containing effluent by immobilization pellets in the device.

Our engineering yeast can adsorb the nickel-containing effluent of 15mg/L to only about 0.5 mg/L in 45 minutes, which basically meets the national discharge standard of nickel ion, and our method has the advantages of high efficiency and low cost.

Improvement of nikABCDE

Two kinds of engineering bacteria were cultured in 30 mg/L nickel ion solution. Within 30 minutes, the original bacteria (nikABCDE) reduced the concentration of nickel ions from 30 mg/L to 15 mg/L, but the improved engineered bacteria (His-Tag-nikABCDE-His-Tag) reduced the concentration of nickel ions from 30 mg/L to 10 mg/L. It can be seen from the figure that the improved engineered bacteria has a stronger nickel adsorption ability than the original bacteria.

Fig 6. Adsorption curve of Ni2+ (30 mg/L) by two kinds of engineering bacteriat with time.

Characterization of two EYFP proteins

1. We successfully connected the gene fragment between the promoter and terminator in pSB1C3 and send it for sequencing. The result showed that we correctly connected the gene fragment.

2. The protein expression of E0032, E0034 and blank control in E.coli cells were detected by laser scanning confocal microscope analysis.

The results indicated that both E0032, E0034 and blank control had fluorescence, but the fluorescence is very weak.

3. In order to confirm the expression of these two yellow fluorescent proteins. Another fluorescent protein, green fluorescent protein was ligated in the same vector, with the same promoter and terminator. The fluorescent microplate reader was used to measure the fluorescence of the three bacteria.

From the curve we can see that using these two promoter and terminator, the expression levels of these two yellow fluorescent proteins are very low, lower than the green fluorescent protein, slightly higher than the blank bacteria. We are not sure if other promoters and terminators can express higher values.


Conclusion

We can see fluorescence signals under the laser scanning confocal microscope, which tells that we have correctly built the plasmid. However, comparing with GFP, using the same promoter and terminator, E0034 and E0032 has a very low level of expression. Other promoters and terminators are not tried, this conclusion is only based on the results of this experiment.

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