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Team:Dundee/Project/DetectorPKRC
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Revision as of 16:27, 14 August 2013
The Detector
Aim: To engineer the E. coli EnvZ sensor kinase to respond to microcystin
The EnvZ system is a signal transduction system composed of two parts and is, therefore, described as a two-component regulatory system. Part 1 is the sensor kinase protein located in the membrane of the cell and Part 2 is the response regulator protein. The native EnvZ sensor detects changes in osmolarity.
EnvZ sensor kinase
The sensor kinase EnvZ detects a signal from the environment and auto-phosphorylates. The phosphoryl group is then transferred to the response regulator OmpR. OmpR is a DNA-binding protein.
E.coli is a gram-negative bacteria which means that is has both an inner and outer membrane. The EnvZ sensor sits on the inner membrane (Fig 3).
EnvZ sensor to detect microcystin
What we want to do for our project is replace the periplasmic domain of EnvZ with the PP1 protein (Fig 4). We hope that when microcystin binds to PP1 then it will activate the receptor.
This will lead to the phosphorylation and activation of the DNA binding protein OmpR. We will also express in our engineered bacteria a DNA construct encoding GFP that’s expression is under control of the OmpR protein.
So our cells will turn green in the presence of microcystin and in this way act as a microcystin detector.
Figure 3. .
Figure 4. .
Progress so far...
WSo far we have successfully cloned the N-terminus with PP1 and we are in the process of adding on the C-terminus. We have also found an OmpR regulated construct in the distribution kit and we have transformed cells to make more of this part. We have also identified GFP in the kit and we will try and join these 2 parts together to make our reporter construct.
Characterisation of our receptors
We will be looking to quantify how many of our PrkC receptors are expressed on the surface of the spores and also how many EnvZ sensors are present on our E. coli cells.
We will be able to get hold of some microcystin and we can use this to bind and activate our receptors. We will then measure the amount of fluorescence by flow cytometry or microscopy. We can then quantify the expression of GFP in relation to how much microcystin is presented to our cells. By using values for how many receptors we have on each cell we can calculate the efficiency of our detectors and hopefully use all this information in order to quantify the effectiveness of our detector.
