Difference between revisions of "Team:EPFL/Results"

Activation of CYC1 promoter the ON state

Scaffold RNA based modules seem to be more efficient than both CRISPRa/i and dCas9 fused systems; namely, MS2 scRNA recruitment of VP64 strongly activates gene expression in eukaryotic cells (Zalatan et al., 2015).

Since the CYC1 promoter (BBa_K1723022) activation has never been tested before using the scaffold system (scCYC1_c3, BBa_K1941001), we decided to assemble all the parts required to induce its gene expression (Fig. 1A).

The first measurement was done using an overnight plate-reader (Fig. 1B). We observed that cells transfected with all the components of the activation module express two times more GFP than the negative control. To confirm these results further, we performed another experiment using FACS (Fig. 1C).

First scRNA repression in yeast Tef1 promoter

The endogenous promoter Tef1 was shown to be repressed in yeast by fusing dCas9 with the strong chromatin remodeler Mxi1 (Gilbert et al., 2013). In light of this finding, we designed a repressor system based on a scRNA carrying the well-characterized viral RNA sequence PP7 (scTef1_PP7, BBa_K1941001). The sequence PP7 is recognized by the PCP RNA-binding protein (Chao et al., 2007) at which we fused the transcriptional repressor domain Mxi1 (Fig. 2A). Furthermore, we made another version of scTef1_PP7, equipped with an additional hairpin that recruits a second repressor module (scTef1_2PP7, BBa_K1941002).

To our knowledge, we are the first to show that repression in yeast could be also achieved using scaffold recruiter elements. The results obtained confirmed that our system behaved like a functional transcriptional repressor module (Fig. 2). Additionally, we observed that scTef1_2PP7 did not repress Tef1 promoter more efficiently than scTef1_PP7. We therefore used the simpler system with the single hairpin PP7 in further experiments.

Proof of concept: the inducible NOT gate

After successful repression of Tef1 promoter using scTef1_PP7 (Fig. 2), we wanted to see if we could go even further: why not try to build a full biological gate? Would it be possible to assemble a gate induced by a molecule other than gRNAs (Gander et al., 2016)? We decided to assemble an inducible biological NOT gate. This gate would consist of four basic parts (Fig. 3A): CYC1 promoter (BBa_K1723022), the repressor fused protein PCP-Mxi1, the scaffold scCYC1_PP7 (BBa_K1941003) and dCas9. The input will be galactose and the output will be GFP (Table 1).

The NOT gate timing

Every system that takes an INPUT and returns an OUTPUT is characterized by a “time response”: an interval of time for which the response is transient i.e. it is not at steady state. In electrical engineering, the transient state of the system remains for a very short time while steady-state is the stage of the system as time t approaches infinity. We can extrapolate this definition to biological systems provided that we change timescale. Infinity would mean intuitively overnight or at steady state, but what would the transient state represent ? How long would this temporary state last ?

The FACS data (Fig. 4) gave us a first intuition about the evolution of the system: the OUTPUT of the NOT gate switches between the 1 and the 0 in overnight culture with 2% galactose; however, after just five hours of galactose induction the GFP signal coming from the cells is shifted.

Activation vs repression: a powerful tool to make other gates

The inducible NOT gate was the one of the major milestone of our project. However, scientific curiosity always pushes matters to the limit. The next step would be to design other gates. For now we obviously did not have time to reach this goal, nevertheless we built the foundation for it: we assessed the efficiency of activation and repression in the same transfected cell. For a robust design of new gates, we would need a repression whenever both repressor and activator are present in the same cellular context. Indeed this is what we observed (Fig. 5).
The results obtained confirm our hypothesis that repression prevails over activation: when dCas9 expression is induced by galactose in a cell containing both activator and repressor modules, the GFP expression decreases. This is a crucial validation that our system can evolve in order to build complex gates, which need the property that repression gains when both activator and repressor modules coexist. This information is also fundamental for the implementation of the biological gates in CELLO.

Characterization of CYC1 promoter by scRNAs: plan for the future

Here we resume all the data collected about the regulatory elements of CYC1 promoter.

Inducibility

Biological circuits need to be stimulated by specific inputs, especially when they are used in the context of a biosensor, in order to return an output and pass along the appropriate information. As such, inducibility is a key feature of Intelligene: by creating a NOT gate induced by galactose, we have a straightforward way to demonstrate a situation in which a specific output is dependent on a specific input. In this case, we chose an input which was appropriate for the system we were working in, since natural switches using galactose as an input already exist in yeast. These switches cause yeast to change their metabolism and genetic expression in the presence of galactose. In this experimental section, we aimed to phenotypically change the expression of two different reporter genes in a same population of cells. Moreover, we chose to work with the inducible promoter GAL1 and the constitutive promoter TDH3.

In our design, TDH3 is constitutively active and it produces red fluorescence protein (RFP). When galactose is introduced the expression pattern changes. This leads the GAL1 promoter to drive the expression of both GFP and a gRNA that inhibits TDH3 by CRISPRi. As a result of the galactose induction, cells change phenotype switching from RFP production to GFP production.

Unfortunately, due to time constraints, we did not managed to integrate all the plasmids carrying the parts needed to perform this experiment.