OVERVIEW

Our associative learning circuit requires a sensor that can detect simultaneously occuring signals, in this case nitric oxide and AHL (Figure 1). Such a sensor with these specific inputs has not been described in the literature before. Thus we generated a collection of AND gates responsive to NO and AHL in order to find a design that best fits the requirements of our system.

We also wanted to demonstrate the flexibility of our system and created one more AND gate responsive to lactate and NO. The lactate portion of the AND gate was based on the work done by ETH Zurich 2015 team.

Figure 1: The learning circuit requires simultaneous detection of nitric oxide and AHL. We designed a collection of AND gates for this purpose, using a combination of the NorV promoter and esaboxes.

Components of the AHL-based AND gate

NorV promoter (PnorV) is the native promoter controlling the nitric oxide reduction operon (norRVW) in E. Coli. [1]. It's transcriptional regulator, NorR, can bind nitric oxide and activate gene expression. We used this promoter in combination with basal NorR production in E.coli as the Nitric Oxide (NO) sensor of our AND gate. The PnorV promoter can also be found among the biobricks we submitted to the registry Part:BBa_K2116002.

EsaR (Part:BBa_K2116001) is a transcriptional regulator of the Pantoea stewartii quorum sensing system. [2] In the abscence of 3OC6HSL it can bind DNA and inhibit transcription. An esabox is a 18bp sequence where EsaR can bind. Unlike other quorum sensing regulators EsaR acts as a transcriptional repressor and not an activator. We took advantage of this property, and placed esaboxes either;

i) as roadblocks after transcription start site of PnorV, preventing the polymerase from advancing or;

ii) within the PnorV to establish competitive binding between NorR and EsaR or RNA polymerase and EsaR.

Design Considerations

i) Roadblock AND Gates

This collection of AND gates each have either one, two or three esaboxes placed as roadblock. Since more than one EsaR binding close to each other could create steric hindrance, we constructed variants where the spacing between the esaboxes is either 8bp or 15bp.

System Design	Biobrick	Comments
	BBa_K2116004	With just one esabox as a roadblock, this promoter is relatively leaky.
here I will add all the biobricks we submit.

ii) Competitive Binding and/or Roadblock AND Gates

Here our aim was to prevent either NorR or the sigma54 factor of RNA polymerase from binding the promoter. There are 3 NorR binding sites, and one sigma54 binding site on the NorV promoter (Figure 2). We placed esaboxes either right before the sigma54 binding site, as a replacement for part of the sigma54 binding site, or upstream of one of the NorR binding sites. For one of these designs, we also have a variant where an additional esabox is placed as a roadblock. This was designed as a solution to potential problems with leakiness of the AND gate.

Figure 1:Structure of the NorV promoter. Based on this structure, we decided to place esaboxes in different regions in order to prevent either NorR or the RNA polymerase from binding the promoter before EsaR was released.

System Design	Biobrick	Comments
	Thanks to the sponsors that supported our project: