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 persons fell to love instantly. In this project, we will recreate the love story of "Romeo and Juliet". [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"

The story that we reproduce is divided into four scenes.

(Scene 1) Romeo and Juliet encountered and they fell in love with each other.

(Scene 2) However, providence lane person, this period of pure love might never broom since they were born in two feuding families. In order to keep their relationship, Juliet planned to pretend die by drinking the drug that will put her into a death like coma so that when she woke up there would be no obstacle between their great loves.

(Scene 3) However, this news was not conveyed to Romeo in time. Romeo misunderstood Juliet’s death and committed suicide.

(Scene 4) When Juliet woke up, what waiting for her was not a beautiful future but Romeo’s death. Juliet felt so sad and she followed Romeo by stabbing 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 story 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.

First, to represent “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 a stop of 3OC12HSL production. 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, and the production of 3OC12HSL is stopped.

Third, to realize “Scene3 Romeo’s suicide” in response to Juliet’s deathlike sleep, we designed 3OC12HSL dependent LacI inverter. When Juliet cell is in deathlike sleep, supply of 3OC12HSL is stopped. 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 3OC6HSL dependent LacI inverter. 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

[Cell-cell communication]

[3OC6HSL-dependent_3OC12HSL_production]

positivefeedbackassay19

positivefeedbackassay20

Ⅰ-3 Modeling

fig1,Modeling

To build our cell-cell communication system, we have constructed and characterized several important parts and modules by experiments. However, it is unconfirmed whether E.coli can play all the drama completely. To confirm the feasibility of our cell-cell communication system, we conducted the following simulation.

Result1: Whether our circuit can reproduce “Romeo and Juliet”

To confirm the feasibility of the cell-cell communication system, we simulated the system under typical experimental conditions. Figure(1) shows the result of the simulation about time-dependent change of the concentrations of the two signals. The blue line in Figure(1) represents the concentration of Romeo’s signals in the culture and the red line represents Juliet’s.[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.[PHAs production]

Ecosystem

Water-repellent