Team:Valencia UPV/Circuit

Valencia UPV iGEM 2015

Circuit Design

After weeks squeezing our brains looking for a solution for a biological decoder, we finally came out with this elegant circuit structure. Next we will explain how did we get to it and why did we made each design decision.

First of all, remember that our goal was to create a biological decoder that is easy to operate in remote places with low resources. This premise lead us to take the following decisions:

Our chassis should be: (i) easy to multiply, making possible to produce unlimited physical copies of the circuit with little effort in remote places; and also (ii) highly stable, resistant to harsh environmental conditions as drought, cold or heat, in order to effectively protect the information stored in it. Wait!! Natural Selection already designed a system that meets our needs, didn´t it: It is called Plant Seed. Who are we then to question it?
Our circuit should be controlled by light of different wavelengths: Light is and input that does not need extra space and weight. It also gives the circuit the option to be activated remotely. So we choose to base our circuit onto optogenetically controlled parts.
We should be able to produce as many outputs (medicines) as possible using as little light signals (wavelengths) as possible. The only solution was to use sequential pulses of light, and to make the system remember that temporal sequence: then our circuit should have memory!

Figure 1. Plant, light, memory! Three characteristics, just one circuit.

This three characteristics were the foundations of our project development. Then we started to look for the components. First we looked for optogenetic tools and found (i) a red/far red post-translational switch from plants that is activated with red light and deactivated by far red; and (ii) a inducer system regulated post-translationally by blue light from Avena sativa that ceases its activity after a period of time in dark. These were going to be our main contact breaker for input signalling. As a back-up option we also decided to explore the design of a novel violet/cyan toggle switch based on stony coral (Pectiniidae)that could improve the control of gene expression with a new input.

Once the light signalling systems were chosen, we structured the circuit in three levels: the “First Input Modules” (level 1), designed to “sense and process” the first light pulse; the “Second Input Modules” (level 2), designed to “sense and process” the second light pulse; and the “Products Modules” (level 3) that unfold different “parts” of the total information stored in the system depending on the state of the previous level modules.

Product activation (level 3) requires the dimerization of a DNA binding domain (BD) with a transcriptional activation domain (AD), a dimerization that is selectively mediated by light. ADs are constitutive (level1), whereas BDs´ expression is regulated by light (level 2). Of a total of 4 BDs, two of them are produced in response to red light and the other two after blue light exposure. A first pulse of light decides which pair of BDs are produced, generating two branches in the circuit. The second pulse of light decides which of the ADs dimerize with the only two BDs present in the system after pulse 1 decision.

However, in absence of an efficient memory mechanism, when pulse 2 colour is different from pulse 1 colour, the alternative level 2 branch will become activated by pulse 2, activating the production of the two additional BDs and producing an interference. To avoid this, we introduced a new type of circuit components in our design: the “Site-Specific Recombinases”. Recombinases function as “molecular scissors”, cutting away fragments of DNA and creating short-circuits. Each branch in level 2 carries a specific recombinase that short-circuits the opposite branch. In this way, the system keeps memory of what happened during pulse1 (blue or light), and pulse 2 can not longer interfere with level 2 elements.

Figure 2. Diagram of information processing inside the circuit.Level 1 corresponds to the constitutive expression of the first two switches that will allow the circuit to work. If red light is given, figures A and B will interact and the production of E, F and G will be activated (level 2). E and F are the two orthogonal optogenetic domains capable to interact with the constitutive expressed activation domains when the adeccuated light is given. G is the memory of the circuit it eliminates the homologous production of level 2 which would had been activated by blue light. In this way G eliminates the oissible interferences produced in case that the second light pulse to activate level 3 is the opposite of the one given in level 1. In this way the second pulso will activate alpha if it is red and beta if it is blue.

Component	Symbol	Component	Symbol	Component	Symbol
DB1-PIF6		BD3-PIF6		Product α
PhyB-VP16		BD4-LOV2		Product β
BD2-LOV2		ƟC31		Product γ
ePDZ-VP16		BD5-PIF6		Product Ω
BxB1		BD6-LOV2