Difference between revisions of "Team:Tuebingen/Description"

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Revision as of 23:17, 18 September 2015

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The expression of the Dronpa-Cre-Dronpa construct is under control of a promotor, that is influenced by a Sensor. Any Biosensor that is able to positively regulate a promotor can be used in our setting.

After expression the Dronpa-Cre-dronpa construct is consituted of fluorescent dronpa domains that attach to each other and thereby inhibt the Cre domain sterically.

Illumination with violet light (400nm) re-activates dronpa fluorescence and also leads to the closed conformation of the construct.

Illumination with blue light (500nm) deactivates dronpa fluorescence and thereby also multimerisation of the dronpa domains.

In the induced open conformation of the construct, the dronpa domains are only losely connected to the Cre, which thereby becomes active.

The RFP-Luziferase reporter swicth leads to expression of RFP if the memory system has not been activated by CREllumination.

After Activation of the Cre protein it removes the loxp-RFP-loxp part of the reporter switch. This leads to expression of Luciferase, which serves as the final reporter of the system.

Project Description

A Biosensor Memory Module: Cre Sensor

Photostimulation is one of the most important non-invasive analysis methods allowing researchers to examine the relationship between metabolic processes, e.g. through activating a molecule via light treatment. We want to create a module which makes it possible to take a snapshot of the activity of a sensor at any time. Our designed system should be capable of capturing such a ‘snapshot’ of the activity of a sensor. Therefore our aim is to create a Cre recombinase whose activity can be reversibly controlled by light. By activating this construct only for a short period of time, we can use a Cre to switch on expression of Luciferase in only a limited amount of the sensor cells. Through coupling the Cre expression to the sensor we can thereby permanently write the sensor state of a given time point into the DNA of a system.

To achieve the construction of a reversibly activatable Cre recombinase we want to apply the caging mechanism described by Zhou et al [1]. This caging is performed by fusing a copy of a variant of the fluorescent protein Dronpa to both the C- and N-terminus of the Cre recombinase. Since this Dronpa variant is able to form monomers or dimers depending on illumination with light of different wavelengths, we hope that the dimerized form inhibits the activity of the Cre recombinase.

Because our system only needs the expression of the caged Cre construct to be dependent on a sensor, it can be combined with almost all Biosensors that include a means of transcriptional control. This gives the system a wide variety of possible applications, especially in the context of the work of other iGEM teams.

[1] Zhou, XX; Chung, HK; Lam, AJ & Lin, MZ. (2012). Optical control of protein activity by fluorescent protein domains. Science, 338(6108), 810-814.