Ⅰ-0 Abstract

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 enzyme in cell Romeo, and 3OC12HSL is synthesized by LasI enzyme 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, we achieved complete positive feedback system for “Scene1 Fall in love” by construction and characterization of two important parts: Plux-LasI and Plas-LuxI. We confirmed that 3OC6HSL-dependent 3OC12HSL producer cell synthesized enough concentration of 3OC12HSL to induce the Las reporter. In the presence of 3OC6HSL, the fluorescence intensity of Las reporter became 42 times stronger than that of Las reporter in the absence of 3OC6HSL (fig1). We also confirmed that 3OC12HSL-dependent 3OC6HSL producer cell synthesized enough concentration of 3OC6HSL to induce the Lux reporter. In the presence of 3OC12HSL, the fluorescence intensity of Las reporter became 116 times stronger than that of Las reporter in the absence of 3OC12HSL. (fig2). Then, to accomplish the complete positive feedback system, we confirmed that the production of a signal activates the production of the other signal, when both type of cell coexist. GFP was expressed in both Las reporter and Lux reporter only when the supernatant of co-culture in which both 3OC12HSL dependent 3OC6HSL producer cell and 3OC6HSL dependent 3OC12HSL producer cell were contained was used as inducer (fig3).

Second, to accomplish the 3OC6HSL-dependent band detect system for “Scene2 Juliet’s deathlike sleep”, we characterized Lux/Tet hybrid promoter which is important part for our band detect system. Plux/tet-GFP expressed fluorescence only in the presence of both 3OC6HSL and aTc. In this situation, the fluorescence intensity became 521 times than that in the absence of both inducers (fig4).

Third, to represent “Scene3 Romeo’s suicide” and “Scene4 Juliet’s suicide”, we constructed two communication inverters: Plux-LacI and Plas-LacI. Plux-LacI expresses LacI repressor in the presence of 3OC6HSL. On the other hand, Plas-LacI expresses LacI repressor in the presence of 3OC12HSL.

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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&Juliette", 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 will be no obstacle between their great loves.

(Scene 3) However, this new 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 that is waiting for her. Juliet felt so sad and she followed Romeo by stabbing herself with a dirk.

Design of genetic circuit

fig5,gene circuit overview

Pon: promoter which is turned on.

Plux: promoter activated by LuxR/3OC6HSL complex.

Plas: promoter activated by LasR/3OC12HSL complex.

Plac: promoter repressed by LacI.

Plux/tet: hybrid promoter activated by LuxR/3OC6HSL complex and repressed by TetR

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.

fig6,Scene1 positive feedback system

The love between Romeo and Juliet is realized by positive feedback of cell-cell communication signals (fig6). In our positive feedback system, two types of E.coli communicate and regulate each other’s production of signal. An increase of one signal causes the increase of the other signal production, resulting in the increase of the signal itself. For the positive feedback system, two quorum sensing modules, LuxI/LuxR and LasI/LasR, which enable two-way communication, were used. Juliet cell expresses LuxR, which is a 3OC6HSL-dependent transcriptional regulator, constitutively. While Romeo cell expresses LasR, which is a 3OC12HSL-dependent transcriptional regulator constitutively. In cell Juliet, LuxR bound with signal 3OC6HSL, and LuxR/3OC6HSL complex activates the expression of LasI. In the mean time, 3OC12HSL produced in Juliet cell is bound by LasR. LasR/3OC12HSL complex activates the expression of LuxI. LuxI and LasI are signal synthesizing enzymes, so the concentration of signal molecules becomes higher when the transcription of LuxI and LasI is activated. The higher concentration of signal molecules, the more complexes exist in each cell, and the activation of signal senders gets stronger. As a result, the increase of the signal synthesis is accelerated.

fig7,band detect system

The “Juliet’s deathlike sleep” that is represented by the stop of 3OC12HSL production is realized by 3OC6HSL-dependent band detect system (fig7). The band detect system is composed of TetR whose expression is regulated by LuxR-3OC6HSL complex and LasI whose expression is regulated by LuxR-3OC6HSL complex and TetR. When the concentration of 3OC6HSL reaches the moderate level, LuxR-3OC6HSL complex moderately activates the expression of TetR and LasI. In this situation, the concentration of TetR is not enough to repress the expression of LasI. Thus, Juliet cell produces 3OC12HSL. As the concentration of 3OC6HSL reaches higher level by the positive feedback, the concentration of TetR reaches to enough level to repress LasI expression. As a result, 3OC12HSL production by Juliet cell is stopped.

fig8,communication dependent inverter

“Romeo’s suicide” in response to Juliet’s deathlike sleep is realized by 3OC12HSL-dependent LacI inverter system (fig8). In the presence of 3OC12HSL, LacI whose expression is regulated by LasR/3OC12HSL complex represses the expression of the lysis gene. Thus, Romeo cell keeps alive when Juliet cell produces 3OC12HSL. However, when Juliet cell is in death like coma, supply of 3OC12HSL is stopped and then the expression of LacI is also stopped. In the absence of LacI, the lysis gene is expressed and Romeo cell dies.

fig9,communication dependent inverter

“Juliet’s suicide” in response to Romemo’s suicide is realied by 3OC6HSL-dependent LacI inverter system (fig9). Juliet cell can keep alive in the presence of 3OC6HSL, because LacI whose expression is regulated by LuxR/3OC6HSL complex represses the lysis gene. After the death of Romeo cell, supply of 3OC6HSL is stopped, and the expression of LacI is stopped. As a result, lysis gene is expressed and Juliet cell dies.

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.

Model development

To simulate the cell-cell communication system, we developed an ordinary differential equation model. The equations used in the model are shown in figure(X). [Model development]

figure(X): The equations used in the model

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(X) shows the result of the simulation about time-dependent change of the concentrations of the two signals.

figure(X): The equations used in the model

We verified the time-dependent behavior of the signal concentration by referring to “Romeo and Juliet” scenes. As described below, the behavior of the signal concentration is consistent with the development of the story.

figure(X): The equations used in the model

figure(X): The equations used in the model

In the gray area of Figure(Xa), the concentration of two signals increases. It looks as if Romeo and Juliet fall in love.

In the green area of Figure(Xa), as the concentration of Romeo signals increases to the some extent, the concentration of Juliet signals starts to decline. It looks like Juliet’s deathlike sleep.

In the blue area of Figure(Xa), lysis gene is expressed in Romeo cell in response to the decline of the concentration of Juliet signals, and the concentration of Romeo signals starts to decline. This represents the suicide of Romeo. In the story, he thought Juliet was dead, and killed himself.

In the pink area of Figure(Xa), lysis gene is expressed in Juliet cell in response to the decline of the concentration of Romeo signals, and the concentration of Juliet signals decreases further. As a result, the concentration of two signals forms a pattern of decline. This represents well that Juliet noticed Romeo’s suicide and followed him afterwards.

Result2: Validation of three subsystems’ function

In [result 1], we demonstrated that the behavior of signal concentration is consistent with the “Romeo and Juliet” story. Next, in this [result 2], we confirmed that the behavior of signal concentration is certainly caused by three subsystems’ function. We confirmed the function of three subsystems (Positive feedback system, Band detect system, and Communication-inverter dependent suicide system). In the Positive feedback system, two kinds of signals increase their concentration mutually. In the Band detect system, the repressor protein (TetR) is expressed in the particular range of the Romeo signal concentration. In the Communication-inverter dependent suicide system, the expression of lysis proteins is repressed in the presence of signals and is promoted in the absence of signals.

figure(X): The equations used in the model

figure(X): The equations used in the model

figure(X): The equations used in the model

(1)Positive feedback system

To confirm the importance of the positive feedback in our cell-cell communication system, we simulated the behavior of the systems with and without positive feedback. In addition to the complete cell-cell communication system, we prepared two systems without positive feedback. First, we prepared the constitutively signal producing system. In that system, LuxI (proteins that generate Romeo signals) and LasI (proteins that generate Juliet signals) are constitutively expressed (Figure(XX)). Second, we prepared the separately cultured cells system (Figure(XXX)). In that system, Romeo cells and Juliet cells are cultured separately.

In Figure(X), the behavior of signal concentration in the complete cell-cell communication system is shown. As a comparison, in Figure(XX), the concentration of Romeo signals and Juliet signals increases slightly at first, but starts to decline before rising sufficiently. In Figure(XXX), the concentration of Romeo signals decreases while Juliet signals increases. That is to say, the cell-cell communication system without positive feedback is unsuitable for reproduction of “Romeo and Juliet” and we confirmed the importance of the positive feedback in our cell-cell communication system.

(difference between parameters of two system? : Yasuo)

(2)Band detect system

Next, we confirmed the function of the Band detect system. Figure(X) and Figure(XX) show the concentration change of the output signals responding to the concentration change of input signals.

In Romeo cells, the production of Romeo signals increases monotonically with the increase of Juliet signals.

On the other hand, in Juliet cells, the production of Juliet signals is in the largest quantities under the particular range of Romeo signal concentration. However, the production of Juliet signals is kept in a low level in the situation of low and high Romeo signals concentration.

In this manner, the validity of Band detect system in Juliet cells was proven by modeling.

Incidentally, the first band detect system in synthetic biology was constructed by Ron Weiss et al. in 2005. In that system, there are two repressors, LacI and LacIμ, that have different repression efficiencies for the LacI promoter. The expression of two repressors is suitably controlled depending on the concentration of signal molecules (3OC6HSL), so the LacI promoter is activated under the particular range of the concentration of signals (3OC6HSL). Compared with that system, our new band detect system has a merit. That is to say, our system is constructed with fewer components. In our system, we employ the Lux-Tet hybrid promoter. The promoter is activated by Lux-3OC6HSL complex and is inhibited by TetR protein. Besides, the expression of TetR is activated only in the situation of high concentration of Lux-3OC6HSL complex. In this way, the hybrid promoter is activated under the middle concentration of signals.

(3)Communication-inverter dependent suicide system – in Romeo cell

To confirm the function of the Communication-inverter dependent suicide system in Romeo cell, we examined the relation between the concentration of Juliet signals and the population of Romeo cells.

On the Line(1) in Figure(X), when the concentration of Juliet signals is high (Figure(Xa)), the expression of LacI (proteins that inhibit lysis gene) is promoted strongly in Romeo cells. Thus, the expression of lysis proteins in Romeo cells is inhibited (Figure(Xb)). As a result, the population of Romeo cells increases (Figure(Xc)). On the other hand, on the Line(2) in Figure(X), when the concentration of Juliet signals is low (Figure(Xa)), the expression of lysis proteins in Romeo cells is promoted (Figure(Xb)). Thus, the population of Romeo cells decreases (Figure(Xc)).

In this way, we verified that there was a negative correlation between the concentration of Juliet signals and the expression of lysis proteins in Romeo cells. Furthermore, we confirmed that the increase and decrease of Romeo cells is dependent on the concentration change of Juliet signals. Therefore, it is well shown that the Communication-inverter dependent suicide system in Romeo cell is correctly functioning.

(4)Communication-inverter dependent suicide system – in Juliet cell

Next, to confirm the function of Communication-inverter dependent suicide system in Juliet cell, we examined the relation between the concentration of Romeo signals and the population of Juliet cells.

On the Line(1) in Figure(X), when the concentration of Romeo signals is high (Figure(Xa)), the expression of LacI(proteins that inhibit lysis gone) is promoted strongly in Juliet cells. Thus, the expression of lysis proteins in Juliet cells is inhibited (Figure(Xb)). As a result, the decrease of the Juliet cell population stops. (Figure(Xc)). On the other hand, on the Line(2) in Figure(X), when the concentration of Romeo signals is low (Figure(Xa)), the expression of lysis proteins in Juliet cells is promoted (Figure(Xb)). Thus, the population of Juliet cells decreases (Figure(Xc)).

In this way, we verified that there was a negative correlation between the concentration of Romeo signals and the expression of lysis proteins in Juliet cells. Furthermore, we confirmed that the increase and decrease of Juliet cells is dependent on the concentration change of Romeo signals. Therefore, it is well shown that the Communication-inverter dependent suicide system in Juliet cell is correctly functioning.

Perspective

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