Difference between revisions of "Part:BBa K346005:Experience"
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The three subparts are ligated together step by step with sub-clone. To test the function of the device, both expression experiment and function test is necessary. As a result, we have test the size of the expressed proteins with SDS-page and Western blot. Besides, to test the efficiency of mercury binding, we also carried out the function test with ICP-AES, which can test the quantity of mercury binding by the bacteria with the device.
 
The three subparts are ligated together step by step with sub-clone. To test the function of the device, both expression experiment and function test is necessary. As a result, we have test the size of the expressed proteins with SDS-page and Western blot. Besides, to test the efficiency of mercury binding, we also carried out the function test with ICP-AES, which can test the quantity of mercury binding by the bacteria with the device.
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== Results: ==
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'''Expression of proteins'''
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The Dsba-mbp, mbp and lpp-ompa-mbp are inserted into the commercial plasmid PET21A. Then the plasmid is transferred to E.coli strain BL21, which can generate T7polyerase when induced with IPTG. Both induced cells and uninduced cells(as control) are centrifuged to get the cytosol, the periplasm and the membrane separated. The SDS-page and Western blot of the expressed proteins in these three parts(figure2) show that induced cells expressed an identical IPTG-inducible protein at the proper place with the size of ~12kD for MBP, ~40kD for Dsba-MBP and ~27kD for LPP-OMPA-MBP, all of which are consist with the predicted size, indicating that all these three coding sequence can be expressed normally in the right place.
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[[Image:mercury device figure2.jpg]]
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'''Function test'''
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Having made sure that the protein can express normally in the proper place, the function tests experiment are carried out with ICP-AES. To test the efficiency of mercury absorption of our mercury bioabsorption device in different concentration of mercury, the concentration gradient is set from 10^-7M to 10^-5M, the results are shown in figure 3. It is obvious that the efficiency of this mercury absorption device increases with the increase of the mercury concentration and it can binding Hg(II) with high efficiency and high sensitivity from the concentration of 10^-6 M compared to that of the control. In addition, compare the capacity of metal binding of the device which contains three subparts with the subparts alone(MBP, Dsba-MBP and LPP-OMPA-MBP), these four parts are tested in the mercury concentration of 10^-5M to compare with each other, with the results shown in figure 4. It is necessary to point that that the device consisting of the three subparts seems to be less efficient than that of the surface display part: lpp-ompa-mbp though it is better than the mbp and Dsba-mbp. This “unusual” phenomenon can be explained as that with the number of exogenous protein increases, the efficiency of expression of protein decreases quickly, for the hard burden due to these proteins.
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  [[Image:mercury figure3.jpg]]  [[Image:mercury figure4.jpg]]
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Revision as of 23:31, 24 October 2010

Experiment:

The three subparts are ligated together step by step with sub-clone. To test the function of the device, both expression experiment and function test is necessary. As a result, we have test the size of the expressed proteins with SDS-page and Western blot. Besides, to test the efficiency of mercury binding, we also carried out the function test with ICP-AES, which can test the quantity of mercury binding by the bacteria with the device.


Results:

Expression of proteins


The Dsba-mbp, mbp and lpp-ompa-mbp are inserted into the commercial plasmid PET21A. Then the plasmid is transferred to E.coli strain BL21, which can generate T7polyerase when induced with IPTG. Both induced cells and uninduced cells(as control) are centrifuged to get the cytosol, the periplasm and the membrane separated. The SDS-page and Western blot of the expressed proteins in these three parts(figure2) show that induced cells expressed an identical IPTG-inducible protein at the proper place with the size of ~12kD for MBP, ~40kD for Dsba-MBP and ~27kD for LPP-OMPA-MBP, all of which are consist with the predicted size, indicating that all these three coding sequence can be expressed normally in the right place.


Mercury device figure2.jpg



Function test


Having made sure that the protein can express normally in the proper place, the function tests experiment are carried out with ICP-AES. To test the efficiency of mercury absorption of our mercury bioabsorption device in different concentration of mercury, the concentration gradient is set from 10^-7M to 10^-5M, the results are shown in figure 3. It is obvious that the efficiency of this mercury absorption device increases with the increase of the mercury concentration and it can binding Hg(II) with high efficiency and high sensitivity from the concentration of 10^-6 M compared to that of the control. In addition, compare the capacity of metal binding of the device which contains three subparts with the subparts alone(MBP, Dsba-MBP and LPP-OMPA-MBP), these four parts are tested in the mercury concentration of 10^-5M to compare with each other, with the results shown in figure 4. It is necessary to point that that the device consisting of the three subparts seems to be less efficient than that of the surface display part: lpp-ompa-mbp though it is better than the mbp and Dsba-mbp. This “unusual” phenomenon can be explained as that with the number of exogenous protein increases, the efficiency of expression of protein decreases quickly, for the hard burden due to these proteins. 

 Mercury figure3.jpg   Mercury figure4.jpg




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