Team:FAFU-CHINA/Demonstrate









Demonstrate

The growth curve of wild type

We measured the growth curve of wild type under the stress of lead and cadmium, by measuring the value of OD600.

First, we set up a beef extract peptone medium containing different concentrations of lead (10 mg / L, 100 mg / L, 300 mg / L, 500 mg / L, 700 mg / L, 1000 mg / L)(see protocols for details) and measure the OD600 value every 2 hours to draw the growth curves.







We noted that Bacillus megaterium is under stress and therefore difficult to grow in beef culture peptone containing concentrations of 1000 mg / L of lead. At 100 mg / L of lead concentration, wild type can grow, but inhibited. At 10 mg / L Pb concentration, the growth curve is very close to the wild type.

In order to observe the growth more intuitively, we took 100 μL from every heavy metal stress cultures to spread plate after 24 hours culture.







It can be seen that before the 700mg / L, the number of colonies close to the CK group, indicating that the bacteria have a lot of proliferation. In the case of 1000 mg / L, the number of colonies was significantly reduced, indicating that the bacteria were difficult to proliferate under the heavy metals of the concentration, which is consistent with the growth curve.

Then we used the same method to measure the growth of Bacillus megaterium at different concentrations of cadmium (concentration gradient of 10 30 70 90 100 200 ... .1500mg / LCd2+), also selected 10mg / L 100mg / L 1000mg / L draws the growth curve







We can see that Cd2+ has a significant effect on the growth of bacteria. At 1000mg / L, the growth was similar to that under lead stress, and it was difficult to grow. We also noted that the tolerance to cadmium of Bacillus megaterium is weaker than that of lead. At a concentration of 100 mg / L, the stagnation period was very long and finally began to proliferate after 14 h. Even in the concentration of 10mg / L, the growth curve is also quite different compared with wild type.

We also took 100 μL of culture to spread plate, the results are as follows







It can be observed that at 100 mg / L Cd concentration, the bacteria can grow and proliferate. After 200 mg / L, the number of single colonies decreased significantly, indicating that the strains were difficult to grow at these concentrations, and it was also seen that Bacillus megaterium was more sensitive to cadmium




After the strain was modified

In the absence of any heavy metal stress, we measured the growth curve of strain after transformation







We can see that the growth of wild-type strains is the best, at 24h the OD value is about 1.2, and the transformation of the strain was significantly weaker than the CK group, were close to 1.0. GST-CRS grows weakest because metallothionein inhibits microbial growth. According to the growth curve, the transformed strain was able to grow normally in the beef extract peptone medium, even with a slight decrease relative to CK.

After the Lpp-OmpA-MBP (Pb) and DsbA-MBP (Pb) were transferred to Bacillus megaterium, and cultured them in beef extract peptone medium with lead + xylose for 24 hours. Then we measured the curve in same way.







Under high concentration(1000mg/L) of lead, the growth status of the transformed strain was significantly improved compared with the wild type. Under relatively low concentrations of lead, the growth status of the transformed strain decreased, but it was still acceptable.

After GST-CRS5 was transferred to Bacillus megaterium, the xylose was added and the growth curve was measured by the same method. The results were as follows.







Similarly, under extremely high concentrations of lead, the transformed strain is still difficult to grow, showing that the strain is highly sensitive to cadmium. The growth status of the transformed strain was also lower than that of the wild type under medium and low concentration of cadmium stress, but it is not particularly significant, which indicated that the introduction of GST-CRS5 into the strain could help it grow under low concentration of cadmium.




Increased ability of transgenic Arabidopsis thaliana expressing the ACC deaminase to accumulate Cd

ACC is the immediate precursor of ethylene in plants. The bacterial enzyme ACC deaminase is the only non-plant enzyme that metabolizes ACC; the enzyme converts ACC to α-ketobutyrate and ammonia. In our project’s phyto-route, we planned to use transgenic plant increasing ability of accumulating heavy metals. However, it is relatively hard to construct transformation system of hyperaccumulator——Sedum alfredii Hance, in this regard, we utilized transgenic model plant Arabidopsis thaliana under the control of 35S promoter as the role of phyto-route.





The blue bar is non-transgenic Arabidopsis thaliana and the yellow bar is transgenic Arabidopsis thaliana. Each of the three transgenic Arabidopsis thaliana contained a single copy.

We made of assay from primary root length and fresh weight those two aspects under Cd treatment. The short term effects of the presence of Cd was monitored following 15d in sterile growth plates. For the most part, the growth of the transgenic plants was inhibited to a lesser extent by the presence of the Cd than was the growth of the non-trans-formed plants. From fresh weight graph, we could see the ability of accumulation of transgenic plants are more better than NT.

Under this circumstance, we just could say that in the lab condition, we got ideal results while in real life the conditions are much complex; soil environment, combined pollution and other stresses those effect factors are so complicated to captured.


PSB-plant interactions for absorption of heavy metals

Based on information and data of above experiments, we planned to integrate PSB with plants for cleanup of heavy metals in order to prove the feasibility of our project.Compared with hyperaccumulator, tobacco as a model plant is relatively familiar to us. In this regard, we decided to take 65-70d seedlings that cultivated in the Hoagland's nutrient solution to observe the short-term effect. Under different concentrations of Pb:0, 50 mol/L,100 µ mol/L, 200 µ mol/L, we added WT PSB and engineered PSB(Lpp-Omp-MBP)respectively to verify the impact of GE-PSB by observing the condition of plant growth, We set the blank test of reagent according to the method and each sample makes 3 parallels with 1 plant per pot.

Table 2 The Bioconcentration factor of wild type tobacco in different concentrations of Pb2+

Figure1 The Bioconcentration factor of wild type tobacco in different concentrations of Pb2+

Data analysis

According to the data, it can be seen that the tobacco enrichment coefficient is high at the treatment of pb-10 µ mo1/L, then, all of them are reduced, may be the resistance of tobacco itself is limited, and the addition of PSB ( Lpp-Omp-MBP) make the adsorption of Pb strengthened

The data showed that the enrichment coefficient of tobacco  reached its peak at Pb-10 / mol / L treatment, and then decreased gradually, indicating that the resistance of tobacco itself was limited. With the addition of PSB (Lpp-OmpA-MBP), the ability of tobacco  to adsorb Pb was more significant than that of PSB, and the enrichment coefficient and growth of tobacco  were enhanced.

Take Sedum alfredii Hance seedlings after the cultivation of 6 weeks under the same treatment and repeat the above operation. The difference is that we have added the PSB (GST-CRS5) to further contrast experiments in the Hoagland Nutrient Solution with Cd.

Based on the above-mentioned pre-experiment, we improved the treatment with different treatments: add 12ml PSB and PSB (GST-CRS5) for seven days with different concentration of Cd( CK (0μmol/ L Cd), 50μmol/ L, 100μmol / L Cd, 200μmol / L, 500μmol / L)

Each treatment was repeated 3 times, one plant per pot. After culturing 7d, repeat the operation of pre-experimental and collect the samples.

Results

Table2 The Bioconcentration factor of Sedum alfredii Hance in different concentrations of Cd2+

Figure2 The Bioconcentration factor of Sedum alfredii Hance in different concentrations of Cd2+

Data analysis

On the whole, with the increase of Cd concentration, the enrichment coefficient of Sedum alfredii Hance alfalfa showed the trend of increasing and then decreasing.

Under low concentration stress, CK (no bacteria) had higher enrichment coefficient than PSB or PSB (GST - CRS5). At the same time, acording to the above experiment, we can see that the growth of Bacillus megaterium was better at 100mg / L, and it could grow steadily. Therefore, Bacillus megaterium absorbed the trace Cd by itself, Under the treatment, the enrichment coefficient is lower, that is, under CK, the adsorption capacity of Cd is strong.

According to the above experiment, we can see that with increase of Cd concentration, when Cd was 100μmol / L, the enrichment coefficient of CK was significantly decreased while under the treatment of PSB or PSB (GST-CRS5), the enrichment coefficient of Sedum alfredii Hance was significantly increased, which was significantly higher than CK. It indicates that Sedum alfredii Hance had stronger resistance to stress and higher capacity of the adsorption heavy metal.

Under low concentration stress, CK (no bacteria) had higher enrichment coefficient than PSB or PSB (GST-CRS5). At the same time, acording to the above experiment, we can see that the growth of Bacillus megaterium was better at 100mg / L, and it could grow steadily. Therefore, Bacillus megaterium absorbed the trace Cd by itself, Under the treatment, the enrichment coefficient is lower, that is, under CK, the adsorption capacity of Cd is strong.

In terms of the peak value of heavy metal enrichment coefficient, it can be seen that CK treatment of Sedum alfredii Hance reached the highest at Cd concentration of 50μmol / L, the addition of PSB  treatment reached the highest peak at Cd concentration of 100μmol / L, while the addition of PSB (GST-CRS5) treatment reached the highest peak at Cd concentration of 200μmol / L. It indicates that, compared with the unmodified strain, the optimized strain could significantly improve the stress resistance and enrichment coefficient of heavy metals in Sedum alfredii Hance and all of them showed an increasing trend. When the content of Cd is higher than 500µmol/L, under high concentration of Cd, the enrichment coefficient of Sedum alfredii Hance decreased to a low value, which may be influenced by their own restrictions.

Reference

[1] Zhang Yibin. Research on the Composition and Characteristics of Root Exudate of Sedum alfredii Hance [J]. Zhejiang University 2014