Team:Amazonas-Brazil/Design

Design

Cells are capable of intricate and complex behaviors. One of the main applications of this robust machinery is to engineer bacteria as factories, pumping out therapeutic agents. One of the building blocks to develop safer therapy techniques for living use is the design of biological systems that sense the tumor microenvironment (TME) and activate the production and delivery of a therapeutic agent specifically in cancerous tissue, paving the way to eliminate side effects in other tissues. In order to contribute to solving this major bottleneck, we designed a system to robustly and specifically sense the tumor microenvironment (TME).

SENSOR UNIT

Specificity can be approached by a mathematical perspective as the intersection between a set of elements. Inspired by this logical point of view, our sensor activates the production of a therapeutic agent in the intersection of a set of TME inputs: high lactate and hypoxia.

Logic gates are powerful tools to execute a set of mathematical behaviors. In synthetic biology, AND gates mimics electrical engineering circuits computing the intersection between two inputs.

That is, two elements must come together to perform induction (ON) of the system. Although feasible, this design has limitations: electrical engineering AND gates can be constructed because they’re easily insulated by wires, but that’s not like genetic circuits works. The signals (proteins) aren’t insulated in a sequential way but diffused inside the cell as multiples asynchronous processes running at the same time.

NAND gates are simpler

Instead of thinking genetic AND gates as their electrical engineering correspondents, where two signals (proteins) get together to form a response (inducing a protein of interest), we empowered our design using the intrinsic repressive nature of transcription networks.

In biological circuitry, NAND gates can be easily designed implementing a single repressor-promoter relation, operated using two promoters to control the signal (repressor) production that represses a protein of interest. Interestingly, NAND gates provides a system to design multiple-input AND gates leveraging the same repressor-promoter simply by adding a new promoter.

The main idea behind our design is that the NAND gates are much more simple structures from a biological point of view. We only need to know the relation between a promoter and it’s repressor to make a NAND gate through the regulated production of the repressor by the means of two promoters sensible to environmental characteristics. The creation of the AND gate re-uses the NAND gate previously described, just adding NOT gates to every input.

Because of that, we thought about creating a biosensor specific to sense tumor microenvironment that could be modular and adaptable, so researchers all over the world could easily develop a specific circuit to their needs.

The construction

Our circuit is based on two promoters regulated by fundamental characteristics of cancerous tissues hardly present in healthy tissue: high concentrations of lactate and hypoxia. We implemented orthogonal promoters and repressors to build the NAND gates and the NOT doors (Voigt), which we selected by their steepness (i.e., High Hill Coefficient).

Our design has two fundamental characteristics:

1. Utilizing positive and negative characteristics to generate specificity. One attractive feature is that you can use not only use the presence of some characters but it’s absence to differentiate cancerous from healthy tissues. A promoter sensible to hypoxia can be applied to induce an AND gate, or, a promoter sensible to oxygen can repress the system. You just need to add a repressor directly in your promoter of interest, that will stop the production of the desired molecule when the repressor is functional, this will make it possible to fine tune your system.
2. Adding more than two characteristics make the system less complex. If you desire to add another characteristic to the circuit, you don't need to insert one more AND gate. You can use the modularity of our system and join another promoter-repressor relation to inject a new element. In summary, our design turns possible the use of a modular and multi-input AND gate, modifying the dynamic range of a molecule production by different induction/repression strategies, achieving a greater degree of control.
3. Since our system is modular, the possibilities for combining promoters and repressors, building intrinsic and precise circuits in a fast and easy way becomes a real possibility in view of the principles regarding.

SECRETION UNIT

One important aspect is trying to minimize possible side effects in the human body through our therapy. A secretion system is our idea to create a more reliable and continuous production of the therapeutic protein. The secretion will be performed using the alpha-hemolysin extracellular media export system by the addition of the HlyA sequence to the C-terminus of the gene sequences of interest, allowing fusion of this signal peptide to the protein of interest, enabling its secretion into the extracellular environment, that is, in vivo to the tumor microenvironment.

The HlyA signal peptide is part of the type-1 secretion system (T1SS). This system is composed of three membranes proteins, two that are from the inner membrane and the third is part of the outer membrane, respectively HlyB, HlyD, and TolC. Their assembly form a channel that possibility the externalization of proteins tagged to the HlyA signal.

In our Secretion Device, the paylod that is regulated by the Biosensor Device is tagged to the HlyA signal while the subunits HlyB, HlyD and TolC are constituvely expressed by the promoter J23111. It’s expected that when the Biosensor is in state on, the therapeutic protein is expressed and secreted directly in the tumor.