Difference between revisions of "Team:Tuebingen/Design"

Introduction

In our sensor memory system a light activatable Cre recombinase is used to save the state of the sensor in the DNA. The main challenge we had to overcome for this part of our project, was to find way to control the activity of the Cre recombinase using light. Luckily, we found that by fusing two copies of the protein dronpa to the N- and C-terminus of the Cre recombinase, the protein should become light activatable.

Dronpa

Dronpa is a monomeric fluorescent protein with high similarity to GFP that is derived from the multimeric wild type protein of the Pectiniidae, which is a species of coral [Ando 2004]. Compared to other fluorescent proteins dronpa is of special interest because it shows a photoswitchable behaviour. Illumination of the protein at around 488nm leads to bleaching of the protein inducing a ‘dark state’. Furthermore this transition is reversible, because illumination of the dark state protein at around 405nm recovers almost the full fluorescence potential [Ando 2004].

Recently a mutant version of dronpa was engineered that did not only show photoswitchable fluorescence but also oligomerization behaviour [Zhou 2012] . This mutation is lysine-145 to asparagine and it is located within the beta strand 7 of dronpa, that is part of the oligomerization interface [Mizuno 2008] . Because a change of the fluorophore state within dronpa also affects beta strand 7 [Mizuno 2010] , the oligomerization behaviour of 145N dronpa can be controlled by illumination [Zhou 2012] . This control over quaternary protein structure can be used to control the activity of engineered proteins. Fusion constructs of an enzymatic or effector protein that carry a dronpa protein at both their N- and C-terminus were shown to be inhibited as long as the dronpa copies were fluorescent and olgimerising, but regained their activity upon turning off the dronpa fluorescence [Zhou 2012] .

Cre

The DNA recombinase Cre is a very useful tool for researchers who want to change the genomic makeup of living tools. We choose the Cre recombinase for its ability to cut a DNA region flanked by the loxp recognition sites, which allows us to use this permanent change in the DNA for our memory system. While this application of the Cre recombinase is rather novel, the proteins has been used for years in engineering of genomes especially for the generation of knock out mice [Nagy 2000] . In eukaryotic cells the activity of the Cre protein can be inhibited by preventing it from entering the nucleus up to a certain time point. This can be achieved by fusing the Cre protein to a tamoxifen inducible receptor domain like ERT2 [Graumann 2000] . These variants of the Cre recombinase are very useful for the generation of conditional knock out lines that offer many more options to researchers.

Our goal is to apply the caging mechanism with dronpa as described earlier to the Cre recombinase (compare figure 1). We needed a light activatable protein for our project, because the system should be able to take the momentary state of the sensor and immediately write it into the DNA. This is only possible through light induced activation of the writing process.

Further on a light inducible Cre recombinase would be very useful for researchers working with the Cre recombinase system, e.g. in mouse knockout models. While inducible tissue specific knockouts are already possible , up to now no systems have been described that allow a light inducible or locally limited activation generation of gene knockouts be Cre recombinase activation. We hope that our work will be able to allow the generation of such systems that could be used to study the effects of genes within a cell population or between neighbouring cells.

For the generation of the dronpa caged Cre recombinase we will use biobricks containing the 145N dronpa, wild type Cre recombinase, Delta1-19 Cre recombinase and serine-glycine linkers of different length. After discussions with experts on protein structure prediction (Birte Höcker, MPI für Entwicklungsbiologie) we decided to try out different linkers instead of trying to predict a possibly working solution. During these discussions we noticed that the N-terminal part of the Cre recombinase (AA1-19) seemed to be rather flexible, because it was not part of the crystal structure we looked at . Therefore we decided to also try the caging of a truncated version of the protein, in case the fusion of dronpa to the wild type N-terminus allows to much movement freedom of the protein domains.

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 ) which are flanked by two loxp sites with the same orientation. Behind the second loxp site is the gene encoding for the luciferase (see Figure 1). This setup leads to expression of luciferase only after the Cre recombinase has cut out the RFP-tADH region.

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.