Difference between revisions of "Part:BBa J61032:Experience"

Revision as of 15:08, 4 October 2013

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Please enter how you used this part and how it worked out.

Applications of BBa_J61032

[http://2013.igem.org/Team:ETH_Zurich ETH Zurich 2013] used PhoA in their project as reporter enzyme. To characterize the enzyme they conducted fluorometric assays to obtain Km values. To this end bacterial cells were grown until in exponential growth phase. Upon reaching this, gene expression was induced by AHL (see [http://http://2013.igem.org/Team:ETH_Zurich/Infoproc their system]). After another 4-5h of growth, cells were harvested and lysed, the cell free extract used for the fluorometric assay. The properly diluted CFX was measured on a 96 well plate in triplicates per substrate concentration. A plate reader took measurements at λEx. 365nm and ΛEm. 445nm. The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot™. See the resulting graph below.

Michaelis-Menten-Kinetics of PhoA with 4-MU-phosphate.

[http://2012.igem.org/Team:Technion Team Technion (2012)] have made an assay using this part. The BioBrick they used is BBa_K784000 consisting of T7 RNAP promoter, phoA and T7 terminator.

We have used the part inside a BL21 strain of E.coli which has an endogenouse PET system.
In the presence of IPTG inducer, T7 RNAP is expressed and activates the part promoter which expresses the Alkaline Phosphatase gene (phoA).
The graph below presents the results of the experiment described. The absorbance at 420nm for ‎each of the different concentrations of the inducer in a range of 0.5 μΜ to 1000 μΜ on a ‎logarithmic scale. The absorbance was calculated via different dilutions of the samples: dilutions ‎by 0.5, by 0.25, and by 0.1. The error bars represent the processing of the data collected. The line at ‎the bottom of the graph represents the basal level according to the control result- no IPTG ‎induction.‎

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As can be seen, the graph shows a clear positive tendency- the higher the concentration the higher ‎the absorption, as expected. Starting from a concentration of 40 μM and above, there are only ‎small deviations from the absorption value of 1.2, probably due to the fact that saturation has ‎been achieved. ‎

User Reviews

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 ===Applications of BBa_J61032===
-[http://2013.igem.org/Team:ETH_Zurich ETH Zurich 2013] used PhoA in their project as reporter enzyme. In order to make their mathematical capable of predicting, parameters were needed. Because of that they decided to conduct fluorometric assays to obtain Km values.To this end bacterial cells were grown until in exponential growth phase. Upon reaching this, gene expression was induced by AHL (see [http://http://2013.igem.org/Team:ETH_Zurich/Infoproc their system]). After another 4-5h of growth, cells were harvested and lysed, the cell free extract used for the fluorometric assay. The properly diluted CFX was measured on a 96 well plate in triplicates per substrate concentration. A plate reader took measurements at λEx. 365nm and ΛEm. 445nm.
+[http://2013.igem.org/Team:ETH_Zurich ETH Zurich 2013] used PhoA in their project as reporter enzyme. To characterize the enzyme they conducted fluorometric assays to obtain Km values. To this end bacterial cells were grown until in exponential growth phase. Upon reaching this, gene expression was induced by AHL (see [http://http://2013.igem.org/Team:ETH_Zurich/Infoproc their system]). After another 4-5h of growth, cells were harvested and lysed, the cell free extract used for the fluorometric assay. The properly diluted CFX was measured on a 96 well plate in triplicates per substrate concentration. A plate reader took measurements at λEx. 365nm and ΛEm. 445nm.
 The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot&trade;.
 See the resulting graph below.