Difference between revisions of "Team:FAFU-CHINA/Demonstrate"

Demonstrate

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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

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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.

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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.

Thanks for trangenic seeds support from Prof. Tang

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.

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Wild type tobacc -Pb²⁺

Pre-test

Different treatments for wild type tobacco : The content gradient of different

bacterial liquor was set for 2 strains with Bacilus megaterium when its OD value of 1 and set up 4 differents Cd concentrations: CK ( Pb-0 ), Pb -50 mo1/L ,Pb -100 µ mo1/L, Pb -200 µ mo1/L, Do the blank test of reagent according to the method and each sample makes 3 parallel with 1 plants per pot for 2d

Results

Table 1 The Bioconcentration factor of wild type tobacco in different concentrations of Pb2+

Figure 1 The Bioconcentration factor of wild type tobacco in different concentrations of Pb2+

Figure 1 The Bioconcentration factor of wild type tobacco in different concentrations of Pb2+

Date analysis

On the overall trend, we can see that adding the PSB can promote wild type tobacco adsorption of heavy metals, while indicating that higher concentrations of bacteria can promote wild type tobacco enrichment

The enrichment coefficient of Bacillus megaterium was significantly different at low concentration of Pb-10μmol/L which was the highest peak value of the Bioconcentration factor

It wasn’t consistent with the expected results. Bioconcentration factor of wild type tobacco is low, probably due to the nutrient solution adding Pb2+, which resulting in the concentration of Pb2+ was decreased. We try to replace the nutrient solution of different anions on the Pb2+ precipitation, but after our efforts, ultimately found the improvement effect is not obvious.

Formal experimental treatment

Take wild type tobacco seedlings after the cultivation of 6 weeks under the same treatment and repeat the above operation. The difference is that we added 12ml PSB and PSB ( LPP-OMPA-MBP ) for7d, CK ( Pb-0 ), Pb -50 mo1/L ,Pb -100 µ mo1/L, Pb -200 µ mo1/L, Do the blank test of reagent according to the method and each sample makes 3 parallel with 1 plants per pot

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

Figure2 The Bioconcentration factor of wild type tobacco in different concentrations of Pb2+

Data analysis

According to the data, it can be seen that the wild type 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-OMPA-MBP) make wild type tobacco adsorption pb strengthened

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Sedum alfredii Hance -Cd²⁺

Pre-test

Different treatments for Sedum alfredii Hance : The content gradient of different

bacterial liquor was set for 2 strains with Bacilus megaterium when its OD value of 1 and set up 5 differents Cd concentrations: CK ( Cd-0 ), Cd-50µ mo1/L Cd-100 µ mo1/L, Cd-200 µ mo1/L, Cd-500 µ mo1/L, Do the blank test of reagent according to the method and each sample makes 3 parallel with 1 plants per pot for 2d

Results

Each of the three parallel data is averaged and analyzed as follows

Table 2 The Bioconcentration factor of Bacilus megaterium in different concentrations of Cd2+

Figure2 The Bioconcentration factor of Bacilus megaterium in different concentrations of Cd2+

Data analysis

According to the data, it was found that the enrichment coefficient was low in the Sedum alfredii Hance under different treatments after 2 days, but the enrichment coefficient was increased after adding Bacillus megaterium , which indicated that Bacillus megaterium play a certain role in the promotion of the adsorption of heavy metals.

Compared with adding 6ml of Bacillus megaterium (OD=1), adding 12ml of Bacillus megaterium has higher Bioconcentration factor, when the content of Cd up to 100µ mo1/L. It may be because its limited tolerance in the Holland nutrient solution is high

When the concentration of Cd2+ reached 500μmol / L, the amount of bacteria in the 12ml and other treatment are significantly different, May be the the insufficient amount of bacteria had great influence.

Formal experimental treatment

Different treatments for Sedum alfredii Hance

1、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-MT) to further contrast experiments in the Hoagland Nutrient Solution with Cd.

2、Based on the above-mentioned pre-experiment, we improved the treatment with different treatments: add 12ml PSB and PSB (GST-MT) for7d with different concentration of Cd( CK (0μmo1 / L Cd), 50μmo1 / L, 100μmol / L Cd, 200μmol / L, 500μmol / L)

3、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 Bacilus megaterium in different concentrations of Cd2+

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

Data analysis

Under low concentration stress, CK had higher enrichment coefficient than PSB or PSB (GST –CRS5), and the enrichment coefficient of PSB (GST –CRS5) was higher than PSB,

When the Cd2+ was 100μmol / L, the enrichment coefficient of CK was significantly decreased compare others.

With the optimized treatment, the concentration of Cd increased at the highest peak value the Bioconcentration factor

According to the graph, it can be seen that the enrichment coefficient of Sedum alfredii Hance for 7d was significantly higher than 2d. With the increase of Cd concentration, the enrichment coefficient of Sedum alfredii Hance alfalfa showed the trend of increasing first and then decreasing, which may be influenced by their own restrictions. With the unmodified strain, the optimized strain(with PSB（GST-CRS5) could significantly improve the stress resistance and enrichment coefficient of heavy metals of Sedum alfredii Hance .

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Reference

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