Proof of Concept
When we started the lab project at the beginning of the summer, nobody on the team could imagine the we would end up making something that might actually work. But we had to change our mind! We actually managed to build a biologically inducible NOT gate!
Why all this excitement? One of the goals of synthetic biology is to control the behaviour of living organisms in order to make our life easier. One way to achieve this goal is to create biological circuits within cells, capable of executing complex logic tasks. The NOT gate is the first step to building these complex circuits and is thus the first basic Boolean Operator that we decided to create. However, using Intelligene, we can potentially build any gate. Click here to see the summary of our results regarding our experiments on CYC minimal promoter.
Our NOT gate is unique for many reasons. Firstly, it is built on an innovative system based on scaffold RNAs (scRNAs). Secondly, to our knowledge, our repressor system represents the first successful example of repression in yeast using scRNAs. Finally, the NOT gate is inducible and thus it is an important proof of concept as a first step towards biosensor systems, in which a molecule acts as an INPUT and can switch between ON/OFF states.
In our gate, the INPUT signal is galactose and it switches the system OFF by inducing repression of GFP expression.
Design and Parts
We integrated the following elements into the yeast genome: dCas9 coding sequence (controlled by the inducible promoter GAL1), the reporter gene yeGFP under the CYC1 promoter, the scRNA Cyc1_PP7 and the fused repressor protein PCP_Mxi1. When all the components are present but GAL inducer is absent, dCas9 is not expressed and GFP is produced. On the other hand, when the GAL inducer is present, dCas9 is expressed and it drives the repressor module to the c6 region of the CYC1 promoter. Then, Mxi1 mediates chromatin condensation via histone deacetylase, and the expression of GFP strongly decreases. GFP expression were measured using Fluorescence-Activated Cell Sorting (FACS) and results are reported here.