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OmpN::GFP

In order to validate this inhibition in vivo, Bouvier et al. constructed a translational OmpN fusion to the N terminus of GFP. Figure 3 shows the mentioned OmpN::GFP fusion. They observed that the GFP signal was inhibited by the asRNA, probing that the fusion of the beginning of the OmpN gene with the GFP gene was also regulatable by the RybB asRNA.

Figure 3. OmpN::GFP fusion. The fusion includes the nucleotides -75 to 90 relative to the ATG of OmpN. The beginning of the OmpN CDS is shown in red capital letters. Taken from Bouvier et al., Suplemental Data (2008)

We will take advantage of this strategy by making an analogue fusion of OmpN start sequence with those proteins whose translation we want to regulate. As we can see in Figure 1, the RybB asRNA binds to nucleotides from 5th to 20th relative to AUG. Consequently, we have designed a shorter fusion in which we fuse the first 7 codons of the OmpN gene CDS upstream of the RFP and Sspb CDS. As for the upstream nucleotides of the OmpN gene we have chosen those from the mRNA (see Figure 4) to make sure that no promoter was included. As we can see in Figure 1, these fusions do include the OmpN RBS.

Figure 4. OmpN in vitro RNA sequence. Taken from Bouvier et al., Suplemental Data (2008).

Our final fusions of OmpN::SspB and OmpN::RFP are shown in Figure 5.

Figure 5. Final OmpN::SspB and OmpN::RFP fusions. The OmpN fragment in each fusion is shown in red and the SspB/RFP CDS in black. The complete CDS of our fusion is shown in capital letters, while the upstream UTR, including the RBS, is shown in lower-case letters.

With these fusions, present in the State 2 of our bistable, and expressing the asRNA under the same promoter as in State 1, State 1 machinery will be able to repress State 2 more efficiently (see Figure 6). As in the proteolysis mechanism, we needed bacteria without the RybB asRNA. We have performed the mentioned deletion to our Sspb- and Clpx- strains by using the lambda red protocol (Datsenko & Wanner (2000)).

Figure 6. When State 1 of our bistable is being expressed by temperature (42 ºC), we are not only expressing the LacI repressor of the Lac promoter but we also are expressing the asRNA under the same promoter. This RybB asRNA binds the mRNAs that have the beginning of OmpN mRNA fused, being these SspB and RFP of State 2. When this asRNA binds to SspB and RFP mRNA it inhibits their translation and, therefore, their expression. In conclusion, when the State 1 is active we are repressing State 2 promoter and preventing both State 1 reporter’s and Sspb’s translation.