The Prototype

Our genetic circuit is designed to be activated by red light. This approach is called optogenetics and allows for a wide range of applications that make use of specific wavelengths to trigger a cascade of reactions resulting in user designed outputs.

Genetic circuit in Synthetic Biology Open Language (SBOL) model:

The Sensing Fusion Protein

A fusion protein was designed from Cph1 and NarX proteins. The first, Cph1, was used for its light-sensitive domain which confers light responsiveness to the fusion protein. The second, NarX’s histidine kinase domain, is the first part of the NarX-NarL originally nitrate-responsive Two-Component System (TCS) which activates a cascade of downstream reactions.

The Reporter

Between the two components of the TCS, an amilCP protein was inserted as a reporter. Reporters, which are distributed within specific sites of the circuit, work as 'checkpoints' to verify which steps of the synthesis have been completed.

The Response Regulator

Following amilCP, we inserted NarL, the response regulator of our TCS. The resulting regulator protein is then phosphorylated by NarX to activate the promoter (pNir) for the next section of the prototype.

The Multiple cloning site

The next segment of the prototype begins with a multiple cloning site (MCS) and is followed by GFP as the reporter. The MCS is where genes of interest can be inserted for specific purposes to fit the design of the user. In our case, the MCS is included within the GFP.

Final Checkpoint

After the protein for the function encoded by the MCS is expressed, the last marker is Green Fluorescent Protein (GFP) which serves as the last checkpoint to ensure that the device performed all its functions.

There is a second genetic circuit which encodes for proteins that aid in the synthesis and function of the Cph1 chromophore. More information on this second genetic circuit can be found on this page.

Design considerations:

The Promoter

This prototype has two kinds of promoters: constitutive and inducible. The first part of the main circuit has a constitutive promoter so that all proteins/tools necessary are always present; the second part of the main construct is controlled by pNir, a promoter that responds directly to NarL maintaining the chain reaction and resulting in the gene expression. On the other hand, the vector containing the genes responsible for the chromophore protein is regulated by the same constitutive promoter found in the main construct.

The Ribosome Binding Site and The terminal

In our prototype we use characterized BioBricks as our Ribosome Binding Sites and Terminators. We chose this route because we wanted reliable and well-documented parts for our construct.

Engineering Design Plan

For the hardware section of our prototype, we imagine the design of a portable circular device that is adaptable to the user’s needs. This device would have integrated lights that vary in color for the necessary wavelength. We would activate our prototype and start the bioproduction targeted. The device would be programmed using an Arduino platform and controlled by the user. For this, we envision using a 3D printer to perfect the design of the device. We decided that a portable device would be best because it is more adaptable and user friendly.

Team:UPRM/Design