Team:Tuebingen/Design
Introduction
For our memory system we needed to create a reporter device that will be switched on by our activated writer protein, the Cre recombinase, and permanently retain this status. Because the Cre recombinase is able to cut out DNA sequences that are flanked by loxp sites, we decided to build a reporter cassette that would express RFP only under normal conditions and switch to luciferase expression after activation of the Cre recombinase. We choose the Nanoluc(R) luciferase (designed by the Promega corporation) as final reporter for our system, because luciferase allows an easy readout that is at the same time very accurate. The expression of RFP in the not-activated cells is supposed to ease testing of the reporter device.
General Cre reporters
The design of our reporter cassette is very similar to other Cre reporters used in research: a single promotor is followed by to protein coding sequences, of which the first one flanked with loxp sites, whereby it can be removed from the DNA [Nagy 2000].
Our reporter cassette contains a constitutive promotor (the ADH promotor, BBa_J63005), followed by a RFP gene and the ADH terminator (BBa_E1010 and
Figure1: Processing of the Cre reporter cassette
Our reporter is able to permanently save information, because the Cre induced switch to luciferase expression is irreversible. Without deactivation of the Cre recombinase this process will occur in every reporter within a given cell. In order to relate the luciferase signal of a population of biosensor memory cells, it is therefore necessary that the reporter is only activated within a fraction of the cells. This can be achieved by activating the dronpa caged Cre construct only for a small amount of time, when taking the ‘snapshot’. This leads to a statistically determined reporter activation in some of the cells, because the concentration of of the Cre construct stands in relation to the activation state of the sensor.
On the one side the time frame that is necessary for activation of the reporter, in such a way that an intermediate number of cells is activated, has to be determined empirically for every combination of sensor and promotor, which can be time consuming. On the other hand all this work done to calibrate the CREllumination memory system can also be used in other beneficial ways.
If the interplay of sensor, promotor strength and Cre activation time is well characterised, any one of this factors can be controlled quite easily. For example assume a sensor measuring a very weak signal which can not easily be determined: using a longer activation time of CREllumination system, the strength of the luciferase readout can easily be amplified.