Team:Valencia UPV/Circuit

Valencia UPV iGEM 2015

Circuit Design

After weeks squeezing our brains looking for a solution of a biological decoder we finally came out with this elegant circuit structure. Here we are going to explain you how did we get to it and why did we made each decision.

First of all the idea was to create a decoder but also one capable to be used in places with low resources. This premise gave to the next decision:

Our chassis should be stable in order to protect the information: Seed are the natural selection answer for this porpoise. Who are we then to question it?
Our circuit should be controlled by light: It is and input that do not need extra space and weight. It also gives the circuit the option to be activated remotely.
Our input type should be the same in order to facilitate its usage: then our circuit must have memory!

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

This three characteristics were then our foundations for the project development. Then we started to look for the components. We looked for optogenetic tools and we found a toggle switch activable with red light and deactivated by far red and a blue light inductor which ceases its activity after a period of time in dark. This are going to be our contact breaker for input signaling. However we decided to design a novel violet/cian toggle switch in order to improve the expression control with inputs.

Once the inductors were decided the circuit was structures in three levels: firs input modules, second input modules and products. This structure created two problems to face, as interrupters in the different levels are the same, when the first input is given both first and second levels would be activated. This first problem was solved creating a library of binding domains to DNA and one protein subunit of the switch and their production will be controlled by the first levels switchers. This solution avoid the undesired activation of next levels, but how to control the activation of the previous levels when the second light pulse is given? The solution for this challenge is the usage of recombinases, they will remove the sequence of the products controlled by the switch that has not been activated in the first level.

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