Team:HZAU-China/Processing

Motivation

Many engineered cells that worked well in a laboratory environment didn’t work well in complex natural environments. Natural cell can adapt to the environment since they have a hierarchical regulatory network. So we want to integrate multiple processing functions in our engineered cells. Taking the limited resources in cells into account, we construct one integrated processing module with fewer parts by utilizing the common structures between modules, instead of merely combining different functional modules.

How we design

We put forward a concept of rewirable circuit. The topological structure of regulatory pathway can be modified to adapt to environmental change. First, we constructed a transcription regulatory pathway by combining inducible promoters and corresponding regulators. Besides, we added some special sequences into the synthetic circuit. These sites can be recognized by recombinase protein and help to rearrange the parts connection by site-specific recombination. After the rearrangement, the circuit we designed is rewired to achieve another function.

We use two examples to demonstrate our idea, and we will describe the general steps about how to design rewirable circuits in the modeling part.

In design 1, the processing module is similar to the classic repressilator, which is composed of three NOT gates. cI represses tetR, which represses lacI, which represses cI. What's different is that the transcription directions of these three genes in our design are no longer the same as that in the classic repressilator. This is because we want to put two promoters more closely. And we add a pair of reversed lox sites on either side of the promoters (Fig. 1).

Figure 1. circuit in design 1

After rewiring the circuit, the regulatory relationship among these three genes is altered. It is now cI that represses lacI. So cI and lacI inhibit each other. And the repressilator which is composed of three NOT gates is rewired to be a toggle switch composed of two NOT gates (Fig. 2).

Figure 2. abstract structure in design 1

In design 2, the parts we used are from a quorum sensing system. When the signal molecule AHL binds to luxR proteins, it activates the luxpR promoter and represses the luxpL promoter. LuxI synthesizes more AHL and AiiA contributes to the degradation of AHL. We used luxpR promoter to express luxI and used luxpL promoter to express AiiA (Fig. 3). Then a direct positive feedback loop and an indirect positive feedback loop formed (Fig. 4). When the inversion happens, it’s now luxpR that will express AiiA and it's luxpL that will express luxI (Fig. 3). So now more AHL will repress its own production, and activates its own degradation. And this is a negative feedback loop (Fig. 4).

Figure 3. circuit in design 2

Figure 4. abstract structure in design 2