Revision as of 09:45, 15 October 2018

Design:

There are two possible ways to control the system. One of them is from the outside of the cell via two synthetic signal pathways leading to the production of either an ON or an OFF free gRNA. In these synthetic signal pathways, a signal molecule binds to a receptor, which then releases the specific gRNA. As signal molecules we chose copper, ethanol and estradiol. The reason we chose these compounds is because there are already existing receptor systems (referenz für paper einfügen) and these compounds are rather easy to come by. Copper for exemple is vastly used in organic farming and ethanol is cheap and easily accessible. Also the amount of signal molecules needed to induce the toggle switch should in theory be very small. (stand auch in irgendeinem paper soweit ich weiß) The other way, which we came up with later in the project, is with Liposomes. In this case the ON or OFF gRNA is bound and directly transported into the cell. The reason we chose to introduce a second way to control our toggle switch is that in case one of the receptors doesn’t work at the end of the lab phase, we can still control the toggle switch directly with the gRNA contained in the liposomes, without being dependant on a working receptor. Furthermore, we became aware after a meeting with a plant specialist at our university, that ethanol is highly toxic for plants.

The toggle switch itself is based on a dCas9 system. (It relies on the dCas9 binding to specific areas on the DNA strand and thereby blocking the expression of a certain region.) In case of an ON signal, either by liposomes or by a signaling, the ON free gRNA will bind to dCas9 and direct it to its target sites in the regulatory region of our OFF gene and thereby block the expression. While the OFF gene is blocked, the ON genes can be translated and be expressed. Therefore gRNA can be produced and GFP, our reporter gene, can be expressed. This constant stream of new free ON gRNA will deny the expression of free OFF gRNA even after the original ON signal decays and will therefore hold the system in the ON state. The system will then only switch into the OFF state if an external OFF signal produces an OFF free gRNA which binds to the target sites in the ON gene promoter region and thereby stop the constant flow of free ON gRNA and replace it with its own gene product.

toggle switch von David

As a reporter-system we use a special variety of GFP which has a very short half-life due to an ubiquitin bound to it. This is important because in order to prove that the system can be started and shut off again, the fluorescence of the GFP has to vanish as soon as the OFF signal is given. (Ref. in paper!) If we would use a variety of GFP without ubiquitin, the fluorescence would decay only very slowly and we would have to wait hours or days to prove that the toggle switch is switched OFF. (idee kommt auch von BOKU, also human practices ?)

The chassis for our system are yeast (Pichia pastoris CBS7435) (genau Zelllinie !) and Arabidopsis thalina. We chose Arabidopsis thaliana because its life cycle is very short for a plant (about 6 weeks from germination to mature seeds) and its genome has been fully sequenced. Plus points were also, that there is a scientific group in our building who work a lot with A.t. and who helped us a lot.