We want to establish a way of communicating with eukaryotes and creating a human-organism interaction. We would like to tell an organism when it should perform a certain task, be it the production of a pesticide or the induction of the gene which induces flowering. What all these applications have in common is that we are not forced to change the organism’s environment to get the desired reaction, but instead we tell it how and when to do a certain task. Such a communication platform can create countless new applications in agriculture, research and many other fields.

The goal of our project is to design a genetic mechanism which enables us to switch a gene of interest ON or OFF with a single dose of a signal molecule, similar to switching a lightbulb ON or OFF.

This enables us to define which genes should be expressed at what point in time. Our toggle switch is designed in a way, that any gene of interest can be cloned into the system and toggled with the corresponding signal molecules. There are countless possibilities for genes that could be cloned into such a system, starting from genes which induce the flowering of plants or the production of a certain resistance gene. In our example, the production of a short lived species of GFP is induced or repressed as soon as either the ON or OFF signal molecule enters the cell.

The system is based on a dCas9 toggle switch, which can be activated or shut-down by a single dose of a signal molecule and stays in its state until a different signal is added. The system relies on dCas9 binding in complex with a specific guide-RNA to the respective site in the promoter of a gene and thereby blocking its transcription. This means that the expression of any gene behind this blocked site is being shut down. As signal molecules, we first created receptors for ethanol, estradiol, and copper. Since these receptors are rather complicated to create and represent a critical element in our system we decided to implement another way of inducing the system, which allows us to test the switch without the need of a functioning receptor. We came up with the idea of using guide-RNA, contained in liposomes to activate the toggle switch.