Team:Freiburg/Results

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(Discussion)
(Precipitator)
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'''Precipitator fused with GST tag'''
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===Mathematical modeling===
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Conclusions:
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To determine the Affinity k_D, experiments to find out the binding affinity of the plastic binding domain are necessary. To get a direct access to these values, we cloned the plastic binding domain in front of a GFP. Then, dilution and washing assays could be performed on polystyrene microtiter plates, red out by a fluorescence plate reader.
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The desired parameters could be calculated by measuring dilution rows of GFP proteins and measuring the fluorescence signals at the different concentrations. C_total could be determined by a dilution row with subsequent washing steps, to find out at what [P] concentration there is a saturation. See description of the plastic binding subproject for more detailed explanation on the experimental setup.
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A qualitative experiment to prove that Nickel is binding the Precipitator is sufficient, since k_2 >> 1 and does not play a significant role in our setup. This experiment could have been done using a nanofilter that blocks protein but let through ions. The Nickel concentration of the flow through can then be measured.
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Alternatively purification of the Precipitator by fusing it with a GST-tag could be done, to subsequently measure the absorbance of the protein, before and after adding Nickel to the solution. After Jordan 1974 a detectable change in the absorbance should be detectable after the complex is formed. A similar effect – a colorshift from white to blue -  is visible when one prepares a Ni-NTA column. For this purpose we cloned the GST domain in front of the Precipitator. However towards the end of the project there was no more time to perfom these experiments.
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==='''Precipitator fused with GST tag'''===
The Precipitator was fused with the C-terminus of our GST tag in order to extract and further test the construct for its Nickel binding affinity. We successfully cloned it together using the Gibson Assembly and then subsequently pasted it into the iGEM vector. The submitted sequence was partially confirmed by sequencing.
The Precipitator was fused with the C-terminus of our GST tag in order to extract and further test the construct for its Nickel binding affinity. We successfully cloned it together using the Gibson Assembly and then subsequently pasted it into the iGEM vector. The submitted sequence was partially confirmed by sequencing.

Revision as of 02:34, 22 September 2011


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