Team:SoundBio/Design

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Design
Overview Red Light Circuit Description Circuit Overview Components Cloning Plasmid Design Blue Light Circuit Description Circuit Overview Components Cloning Plasmid Design

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:
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No Red Light

  1. Cph8 is constantly expressed (due to the constitutive promoter)
  2. Cph8 creates a metabolic cascade to phosphorylate OmpR, a protein found natively in the host E. coli
  3. Phosphorylated OmpR is able to bind to the OmpC promoter, causing expression of the dCBD-sfGFP fusion protein
  4. The fusion protein binds to the BC, turning it green

Under Red Light

  1. Cph8 is constantly expressed (due to the constitutive promoter)
  2. Cph8 gets disfigured under the red light, so it can’t start the aforementioned metabolic cascade
  3. OmpR isn’t phosphorylated and therefore, it cannot bind to the OmpC promoter
  4. 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

Cloning

Digestion/Ligation:

Our initial attempt at cloning the Red Light Circuit was to use a simple series of digestion and ligation reactions, because all of the basic parts we were using had compatible BioBrick prefixes and suffixes. However, there was an error in constructing our plasmid, so we were never able to receive it from the company we outsourced it to.

Gibson Assembly:

As the season was growing late, we decided to try again to clone the plasmid. This time, we decided to try Gibson Assembly because it is a fast, scarless assembly method that also would have allowed us to add in our RBS for certain promoters. We designed the plasmids in Benchling using its Assembly Wizard, and then sent the instructions off to the BIOFAB at the University of Washington to get the part actually synthesized for us. However, even though they claimed that this synthesis would be fast, we still haven’t received the part back.

Plasmid Design


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Blue Light Circuit

Description/Purpose

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

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Components

Constitutive Promoter BBa_J23119
Double Terminator BBa_B0015
FixK2 promoter BBa_K592006
YF1+FixJ with RBS BBa_K592016
AmilCP fused to dCBD BBa_K592009

No Blue Light

  1. Yf1 and FixJ are constitutively expressed
  2. The Yf1 pathway leads to FixJ being phosphorylated
  3. The phosphorylated FixJ is able to bind to the FixK2 promoter initiating transcription of the fusion protein dCBD + amilCP
  4. The fusion protein binds to the BC, turning it blue

Under Blue Light

  1. Yf1 and FixJ are constitutively expressed
  2. Yf1 is unable to phosphorylate FixJ
  3. FixJ does not bind to the FixK2 promoter
  4. The fusion protein is not expressed so the BC is not colored red

Cloning

Due to limited time, we were unable to follow through with cloning the Blue Light Circuit. Instead, we shifted the focus of our project onto characterizing only the Red Light Circuit.

Plasmid Design