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<h3 id="PD">Plasmid Design</h3> | <h3 id="PD">Plasmid Design</h3> | ||
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− | <h2 id="BLC">Blue Light Circuit<h2> | + | <h2 id="BLC">Blue Light Circuit</h2> |
− | Description/Purpose | + | <h3 id="D">Description/Purpose</h3> |
Our project focuses on the functionalization of bacterial cellulose which we achieved through light induced expression of chromoproteins. The Blue Light Circuit uses a blue light inducible promoter to regulate the expression of the blue chromoprotein AmilCP. Expression is inhibited by blue light so every part of the BC that remains in the dark will be colored blue. (Source) | Our project focuses on the functionalization of bacterial cellulose which we achieved through light induced expression of chromoproteins. The Blue Light Circuit uses a blue light inducible promoter to regulate the expression of the blue chromoprotein AmilCP. Expression is inhibited by blue light so every part of the BC that remains in the dark will be colored blue. (Source) | ||
Circuit Overview | Circuit Overview |
Revision as of 18:53, 20 October 2019
Design
Overview
Red Light Circuit
Description
Circuit Overview
Components
Cloning
Plasmid Design
Blue Light Circuit
Design Overview
We wanted to use optogenetics to control the functionalization of the BC sheet. To do this, we designed two genetic circuits, each of which are sensitive to a specific wavelength of light, to control protein attachment. Both circuits operate under similar principles: a constitutively expressed light sensor protein that gets phosphorylated under certain light conditions. That phosphorylated protein binds to a corresponding inducible promoter, activating the transcription of a double cellulose binding domain (dCBD) fused to a chromoprotein. The dCBD acts as a linker between the chromoprotein the the BC sheet. So, the end result should be a cellulose sheet with different concentrations of protein attachment depending on light exposure. The two light conditions we chose were red and blue. Their respective circuits are documented below.We also wanted to test that K. rhaeticus could grow under various antibiotic concentrations. We designed a few basic experiments to prove that K. rhaeticus could be cultured with E. coli and that BC could be produced.
Red Light Circuit
Description/Purpose
This circuit controls functionalization based upon red light. Here, the light sensor is cph8 which is a fusion of photoreceptor cph1 and envZ. When there is no red light, cph1/envZ binds to a separate protein complex, chromophore phycocyanobiline (PCB) which causes the phosphorylation of ompR. OmpR-P then binds to the inducible promoter ompC, which activates transcription of dCBD fused to sfGFP. (Source)Because envZ naturally exists in the E. coli genome, this interferes with our circuit. We want the only copy of envZ to be under light control; we can’t have another one floating around that can cause the phosphorylation of ompR. Thus, we used E. coli strain SKA974, which is a JT2-based envZ-deficient strain. (Tabor, 2011)
Additionally, since PCB is not naturally expressed in E. coli we needed to add that in. PCB is composed of two parts, ho1 and pcyA. So for our circuit to function, we also need PCB, which we planned on constructing in a separate plasmid.
Circuit Overview
Red Light System:
PCB:
No Red Light:
- Cph8 is constantly expressed (due to the constitutive promoter)
- Cph8 creates a metabolic cascade to phosphorylate OmpR, a protein found natively in the host E. coli
- Phosphorylated OmpR is able to bind to the OmpC promoter, causing expression of the dCBD-sfGFP fusion protein
- The fusion protein binds to the BC, turning it green
Under Red Light:
- Cph8 is constantly expressed (due to the constitutive promoter)
- Cph8 gets disfigured under the red light, so it can’t start the aforementioned metabolic cascade
- OmpR isn’t phosphorylated and therefore, it cannot bind to the OmpC promoter
- The fusion protein isn’t expressed, so the BC doesn’t get colored green
Components
PCB:
No Red Light:
- Cph8 is constantly expressed (due to the constitutive promoter)
- Cph8 creates a metabolic cascade to phosphorylate OmpR, a protein found natively in the host E. coli
- Phosphorylated OmpR is able to bind to the OmpC promoter, causing expression of the dCBD-sfGFP fusion protein
- The fusion protein binds to the BC, turning it green
Under Red Light:
- Cph8 is constantly expressed (due to the constitutive promoter)
- Cph8 gets disfigured under the red light, so it can’t start the aforementioned metabolic cascade
- OmpR isn’t phosphorylated and therefore, it cannot bind to the OmpC promoter
- The fusion protein isn’t expressed, so the BC doesn’t get colored green
Components
Constitutive Promoter | BBa_J23119 |
---|---|
Double Terminator | BBa_B0015 |
RBS + cph8 | BBa_K592018 |
ompC | BBa_R0082 |
dCBD + sfGFP | BBa_K1321348 |
ho1 | BBa_I15008 |
pcyA | BBa_I15009 |