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Light-Pattern Controlled Circuit

• Overview
• Logic Design
• Workflow
• Results/Modeling
• Future Directions

Logic Design

Before we begin our discussion of the circuit logic, let us first review what must be accomplished by this circuit:

• Input
  • As explained in the Overview section, we desire the input to the cell to be light. Because light cannot alone activate or repress a promoter, the circuit must in some way be able to translate the light signal to a molecule that can regulate transcription. Additionally, using light as input implies a change in light state - from OFF to ON or vise versa. Thus, the molecule that provides the interface between light and the circuit must be able to switch between active and inactive states.
• Processing
  • Given a molecule that toggles between active and inactive, it is easy to imagine a circuit that switches between one state of production and a second. However, we desire to achieve a progression of four states! In order to accomplish this, our circuit, unlike the light responsive molecule, cannot return to its original state when the light turns back OFF. Clearly, our circuit must be able to hold "memory!"
• Output
  • We want our circuit to have distinct states - when possible, we would like a state to turn off after a new state has been turned on (with the exception of the memory, of course!).

Consideration of these requirements clearly gives rise to a handful of modules. The first is the Conversion Module, which takes light ON/OFF and converts it to transcription ON/OFF. This module involves a molecule that is directly modified by the presence or absence of light, and a component that effects a transcriptional change depending on the state of the light responsive molecule.

Second is the Memory Module, which "remembers" that light has been changed in the past. This Memory Module is a crucial way-point in the circuit, as without it the circuit would merely return to its original state when the light turns back OFF.

Third is the IF&NOT Module that generates the third state. As this module can only be active following the first and second states, its activation must depend on the presence of memory ON and the absence of some factor produced only when the light is some α state.

Finally, the last piece of the circuit is the AND Module, which produces the fourth and final state in our circuit. This module uses AND logic because it requires two inputs: one that is only expressed when the third state is active and a second that is expressed when the light is in some β state (the opposite of the previous light state, for the user has inputted a change!).

Contents 1 Light-Pattern Controlled Circuit 1.1 Logic Design 1.1.1 Conversion Module 1.1.2 Memory Module 1.1.3 IF&NOT Module 1.1.4 AND Module

Conversion Module

In order to design the Conversion Module, which converts light ON/OFF to transcription ON/OFF, we looked to past years' iGEM team projects. We found (quite easily, in fact, as it won best new part) team EPF-Lausanne 2009's E.Colight project, which characterized a synthetic protein LovTAP capable of acting as a repressor like tryptophan when exposed to 470nm light. You can read more about their project here. As EPF-Lausanne describes, "Upon light induction, a conformational change is transmitted from the light-sensitive to the DNA-binding domain, which ultimately results in an increase of the regulatory domain's affinity for its respective DNA binding site." This sounds like a match for what we desire! This LovTAP molecule is changed shape under exposure to light, and changes back to its original form when that light is removed. Furthermore, this part has been well characterized. Part of this characterization by the EPF-Lausanne team involved the creation of a read out system, which undergoes a change in transcriptional output in response to the changing conformation of the LovTAP protein under 470nm light. Thus, contained in EPF-Lausanne's work from last year is the essentials of the Conversion Module: <partinfo>BBa_K191003</partinfo> generates the LovTAP protein and <partinfo>BBa_K191005</partinfo> is the response system to transmit the conformational change of LovTAP to a change in transcriptional output. <partinfo>BBa_K191003 SequenceAndFeatures</partinfo> <partinfo>BBa_K191005 SequenceAndFeatures</partinfo>

This system functions as follows: LovTAP is produced (given IPTG is in the culture - this must be changed for the final product to a constitutive promoter) and is expressed to saturation in the cell. When the cell is not exposed to 470nm(light), the TrpR promotor is on, and TetR is expressed, which represses the p(TetR) promotor. Thus, there is no expression the proteins downstream of p(TetR) when the light is OFF. When the light is ON, LovTAP changes conformation to its active form and represses the TrpR promoter. This ceases expression of TetR, which in turn no longer represses p(TetR), and the protein downstream of p(TetR) is expressed. This general system forms the sort of "grand central" of our circuit, and we will revisit it later with the modifications made to it by our team when considering the circuit in whole.

Memory Module

Discuss the specifics of memory module here

IF&NOT Module

Discuss etc.

AND Module

Discuss.