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In order to build dCas9-controlled circuits, we aim to use dCas9-inducible synthetic promoters to mimic the behaviour of a transistor (Go take a look at our design (LINK) if you haven't !). To see whether such promoters could be used in wider-scale circuits, we identified and tested the following desirable properties :

• Transistor response : Is the transistor inducible? Is its output modulable allosterically
• Transistor mutability : Is it possible to mutate the dCas9-targeted sites on transistor-like promoters without altering their behaviour and output levels ? Would it be possible to make a family of homogenous transistors that can be regulated independently?
• Transistor orthogonality : is regulation of a transistor specific? Is there cross-talk? Could several transistors work simultaneously?
• Transistor chainability : Can transistors can be linked serially to allow for multiple levels of information processing ?

Transistor response: BBa_ is inducible. Activation is repressed when both activation and inhibition sites are bound by dCas9-ω

dCas9 binds DNA complementary to its guide RNA (gRNA). The location it binds to may therefore be controlled by producing specific gRNAs.

By targeting dCas9 fused to a RNAP recruiting subunit (dCas9-ω) to an adequate distance from the transcription starting site (TSS), J23117 (BBa_(LINK)) was successfully induced, which represents a PNP transistor in “on” state.

We chose to test two sites close enough to the TSS which when bound by dCas9-w are likely to sterically hinder RNA Polymerase (RNAP). By itself, targeting dCas9-w to inhibition sites (R1, R2), sometimes resulted in slight activation (repeat (LINK)) and sometimes in slight inhibition (repeat (LINK)). This might be explained by the already low promoter strength of J23117 (close to autofluorescence in our measurements and consistent with the registry(LINK)) and the presence of the activating ω subunit on dCas9-ω. Overall, R1, R2 and R1/R2 produce fluorescence close to basal levels and the transistor is considered to be in “off” state.

Yet, when activation and inhibition sites are targeted simultaneously, dCas9-w bound to inhibition position hinders induction (A/R1, A/R2). We found out that A/R2 may virtually suppress activation; the promoter is in “off” state. Such an effect has been reported for S. cerevisiae (REF) but -to our knowledge- hadn’t been tested in E. coli.

The Relative Fluoescence Units (RFU) discrepancy between A/R1 and A/R2 could be explained by our modeling simulations (LINK). A/R2 produces an output consistent with predictions for a model where activation and inhibition sites may be bound by dCas9-w without steric hindrance between the dCas9-ws: binding is independent. A/R1 on the other hand behaves as if co-binding of the activation and inhibition sites were impossible. Interestingly, we notice that for A/R1 the activation and the inhibition sites are on the same strand, whereas in A/R2 the sites are a bit farther and on different strands. If you are interested, please take a look at the promoter modeling (LINK) page for a plausible explanation.

References

[1] Farzadfard, F., Perli, S. D., Lu, T. K. (2013). Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas. ACS Synth. Biol., 2 (10), pp 604–613.