Team:Technion/Project/YES gates

From 2012.igem.org

What's a YES gate?

A yes gate is one of the simplest molecular logic gate operations. The truth table for a YES gate is:

Input	Output
1	1
0	0

Figure 1. a schematic of our YES gates. The Inducer triggers the expression of the FP and RNAP from PI.

The YES gates in our project consisted of an inducer which triggered the expression from a dedicated promoter. Consequentially, a fluorescent protein and an RNA polymerase are produced. The general scheme of a YES gate is presented in Figure 1.

When planing a complex system, there is no way of knowing which combination of promoter and RNAP will work well. Therefore, we planned to attempt the cloning of different RNAPs under the various promoters.
The choice of FPs to follow each promoter was not random. The different considerations will be described below.

YES gate #1

The first YES gate consists of a P_TetO promoter which is repressed by TetR. The expression is induced by anhydrotetracycline (aTc). At first, we attempted the cloning of mCitrine downstream to P_TetO in plasmids we got from Roee. These showed no fluorescence when tested in the plate reader. Therefore, we turned to the registry for alternatives. We found [http://partsregistry.org/Part:BBa_I13522 BBa_I13522] and [http://partsregistry.org/Part:BBa_I13600 I13600] which express P_TetO+GFP and P_TetO+CFP respectively. Restriction analysis of [http://partsregistry.org/Part:BBa_I13522 BBa_I13522] gave us some weird results, so we decided not to use it. Eventually, we decided to use a P_TetO+mCherry from Roee, which he is working with regularly.
The next step was cloning the different RNAPs downstream to mCherry. Unfortunately, we didn't get a working clone with any of the RNAPs in time for the BioBrick submission deadline.

YES gate #2

The second YES gate consists of P_Lac/Ara promoter which is induced by IPTG and arabinose in the presence of LacR and AraC respectively. We got this promoter from Roee followed by the Cerulean gene. This plasmid was used as the starting point for the assembly of YES gate #2. The first step was the addition of a [http://partsregistry.org/Part:BBa_B0015 terminator BioBrick] along with additional restriction sites for the cloning of different RNAPs. However, this cloning step kept failing in several attempts and was abandoned due to lack of time.

Figure 2. A schematic represantation of YES gates #1 and #2

YES gate #3

The third YES gate consists of the P_Lux promoter and a theophylline induced riboswitch (RS). In this YES gate, there is actually a two step regulation. The transcription from the P_Lux promoter is induced by [http://partsregistry.org/3OC6HSL 3OC₆HSL]. The translation is regulated by the RS and is induced by theophylline.
The RS was originally developed in fusion to DsRed Express {1}, which is a red FP from which mCherry was engineered {2}. Since the coding region has effect on the RS function, we thought mCherry ([http://partsregistry.org/Part:BBa_J06504 BBa_J06504]) would be a good candidate. However, the well that was supposed to contain [http://partsregistry.org/Part:BBa_J06504 BBa_J06504] contained some other DNA. As a result, we used PCR to get mCherry from [http://partsregistry.org/Part:BBa_J06702 BBa_J06702]. Unfortunately, using mCherry turned out to be a bad choice, since the regulation by the riboswitch failed. The chronicles of the cloning and experimentation with the RS are described in the RS section.
Since the RS regulates the translation of a protein that is fused downstream to it, it had to be fused not only to the FP but also to the RNAPs. This step was completed successfully for several RNAPs as described in the RS section. However, due to lack of time, we didn't manage to clone the RNAPs downstream to the mCherry in the composite part.
Finally, for this YES gate to work, the P_Lux promoter requires the presence of the LuxR protein. We planned to clone [http://partsregistry.org/Part:BBa_I0462 BBa_I0462] which includes an RBS, the LuxR gene and a terminator downstream to our RNAP (or downstream to mCherry for intermediate testing) as the final part in the assembled YES gate. However, in the 2012 distribution kit, [http://partsregistry.org/Part:BBa_I0462 BBa_I0462] turned out to contain DNA that does not match the expected sequence (wasting valuable time for us). Consequentially, we tried to amplify [http://partsregistry.org/Part:BBa_I0462 BBa_I0462] from [partsregistry.org/Part:BBa_F2620 BBa_F2620]. The PCR failed since the primer tail contained the BioBrick RFC10 suffix, resulting in a primer that annealed to the suffix in the plasmid instead of the end of [http://partsregistry.org/Part:BBa_I0462 BBa_I0462]. Eventually, due to lack of time, we dropped the attempts of assembling the complete YES gate and focused on characterizing the RS as described in the RS section.

Figure 3. A schematic representation of YES gate #3

References

1. Lynch S. A., Gallivan J. P. 2009. A flow cytometry-based screen for synthetic riboswitches. Nucleic Acids Research 37(1): 184-192.
2. Shaner N. C., et al. 2004. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22(12): 1567-1572.