Project overview

A love story contains several processes. Two people fall in love and their love burning wildly. However, no forever exists in the world, in most occasions, love will eventually burn to only a pile of ashes of the last remaining wind drift away. In our project, we have recreated the story of "Romeo and Juliet" by Shakespeare vividly by two kinds of Escherichia coli cell-cell communication. We aim to generate a circuit involving regulatory mechanism of positive feedback rather than commonly-used negative feedback to control the fate of E.coli by signaling between two types of E.coli. Besides, Rose represents love. We are the first iGEM group ever to synthesize PHA (a kind of bio-plastics) by using our new biobrick part, representing rose .

Ⅰ cell-cell communication

"Romeo and Juliet" is the dramas by dramatist William Shakespeare of England. The stage is city Verona, Italy in the 14th century. Romeo met Juliet. Two people fell in love instantly. In this project, we will recreate the love story of "Romeo and Juliet". [Detailed descriptions for Cell-cell communication]

Ⅰ-1 story

We make our cute E.coli play “Romeo and Juliet” which is one of Shakespeare’s most famous plays. In this project, we define the signal that E.coli produce as the romantic feeling of Romeo and Juliet. In this project, we will recreate the love story of "Romeo and Juliet", by using "Cell-cell communication"

Fig1-1,the story of “Romeo and Juliet”

The story that we reproduce is divided into four scenes(Fig1-1).

(Scene 1) Romeo meets and falls in love with Juliet. Once the love between two people stimulates each other, they become deeply attached and cannot live without each other.

(Scene 2) However, Juliet knows that their love will not be accepted by society because of family feud. To keep their relationship, Juliet plans to pretend to be dead. She takes a sleeping potion that makes her fall into a deathlike sleep.

(Scene 3) Romeo has heard of Juliet’s death without knowing the fact that Juliet is alive. Romeo decides to commit suicide by taking poison in response to Juliet’s “deathlike sleep”.

(Scene 4) Juliet awakes to find Romeo dead beside her. She decides to commit suicide in response to Romeo’s suicide. She stabs herself with a dirk.

We designed a cell-cell communication system that makes two types of engineered E.coli play “Romeo and Juliet”. We represented the four scenes with concentration of signal molecules 3OC6HSL and 3OC12HSL. 3OC6HSL is synthesized by LuxI in Romeo cell, and 3OC12HSL is synthesized by LasI in Juliet cell. To reproduce the four scenes, we designed three subsystems: positive feedback system, band detect system, and communication-inverter dependent suicide system.

First, to represent the condition that their love stimulates each other in “Scene1 Fall in love”, we designed a positive feedback system in which the production of a signal activates the production of the other signal.

Second, we applied a 3OC6HSL-dependent band detect system to represent “Scene2 Juliet’s deathlike sleep” by the stop of 3OC12HSL production in Juliet cell. When the concentration of 3OC6HSL reaches higher level by the positive feedback, the concentration of TetR is enough level to repress the expression of LasI, As a result, the production of 3OC12HSL is stopped though cell Juliet is alive.

Third, to realize “Scene3 Romeo’s suicide” after Juliet fell into the deathlike sleep, we designed communication-inverter dependent suicide system in Romeo cell. When Juliet cell is in deathlike sleep, supply of 3OC12HSL is stopped though Juliet cell is alive. In the absence of 3OC12HSL, the lysis gene is expressed and Romeo cell dies.

Finally, to realize “Scene4 Juliet’s suicide” in response to Romeo’s suicide, we also designed communication-inverter dependent suicide system in Juliet cell. After the death of Romeo cell, supply of 3OC6HSL is stopped, and the expression of LacI is stopped. As a result, the lysis gene is expressed and Juliet cell dies.

Ⅰ-2 Positive Feedback of cell-cell communication

Fig1-2,positive feedback assay

To accomplish the positive feedback system in the cell-cell communication, we designed and constructed two types of genetically engineered E.coli, 3OC6HSL-dependent 3OC12HSL producer cell (Plux-LasI cell) and 3OC12HSL-dependent 3OC6HSL producer cell (Plas-LuxI cell). By co-culturing Plux-LasI cell and Plas-LuxI cell, higher concentration of a signal than initial conditions was detected through the production of the other signal (Fig1-3 red arrow & blue arrow). This result strongly suggests that our positive feedback system worked accurately. In the process of the implementation, we also confirmed two modules, the 3OC6HSL-dependent 3OC12HSL production module and the 3OC12HSL-dependent 3OC6HSL production module, that constituted our positive feedback system. In these module, two new Biobrick parts, Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]) and Plas-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934012 BBa_K934012]) , were characterized their functions. [Detailed descriptions for Cell-cell communication]

Fig1-3,[see more]

Fig1-4,[see more]

Fig1-5,[see more]

Ⅰ-4 Modeling

Fig1-6, time-dependent change of the concentrations of the two signals.

Fig1-7: the story of “Romeo and Juliet”

Result: Whether our circuit can reproduce “Romeo and Juliet”

To confirm the feasibility of the cell-cell communication system, we developed an ordinary differential equation model and simulated the system under typical experimental conditions. Fig4-3 shows the result of the simulation. As described below, the behavior of the signal concentration is consistent with the development of the “Romeo and Juliet” story.

In the gray area of fig4-3(a), the concentration of two signals increases. It represents Scene1.

In the green area of fig4-3(a), as the concentration of Romeo signals increases to the some extent, the concentration of Juliet signals starts to decline. It represents Scene2.

In the blue area of fig4-3(a), lysis gene is expressed in Romeo cell in response to the decline of the concentration of Juliet signals, then the concentration of Romeo signals starts to decline. It represents Scene3.

In the pink area of fig4-3(a), lysis gene is expressed in Juliet cell in response to the decline of the concentration of Romeo signals, then the concentration of Juliet signals decreases further. It represents Scene4.

[Detailed descriptions for Modeling]

Ⅱ PHAs Production

Ⅱ-1 Story

There is what of the famous scene of "Romeo and Juliette"

we will recreate the rose come out in the lines of the famous drama "Romeo and Juliet" by the synthesis of PHAs.

Ⅱ-2 PHAs production

fig1,

We made a new biobrick part and succeeded synthesizing Polyhydroixyalkanoates(PHAs). This is the first Biobrick part to synthesize PHAs. In our project, we also draw rose silhouette to produce the balcony scene of “Romeo and Juliet” by the synthesis of PHAs.[Detailed descriptions for PHAs production]

Ecosystem

Water-repellent

Human Practice