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 new signal-producing 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]). With the induction of 3OC6HSL, the fluorescence intensity of the Las reporter cell increased by 20-folds (Fig2-1-1-1). While the fluorescence intensity of the Lux reporter cell increased by 112-folds with the induction of 3OC12HSL (Fig2-1-1-2). By co-culture of two cell types each respectively with one of the signal-producing parts, we then confirmed accomplishment of the complete positive feedback system where the production of a signal activates the production of the other signal. We think this is the most important results in our project. The co-culture contained both 3OC12HSL-dependent 3OC6HSL producer cell (Plas-LuxI cell) and 3OC6HSL-dependent 3OC12HSL producer cell (Plux-LasI cell) (Fig2-1-1-3). By co-culturing Plux-LasI cell and Plas-LuxI cell, we confirmed higher concentration of a signal than initial conditions was detected through production of the other signal (Fig2-1-1-3 red arrow & blue arrow). This result strongly suggests that our positive feedback system worked accurately.

Second, to accomplish the 3OC6HSL-dependent band detect system for “Scene2 Juliet’s deathlike sleep”, we characterized Plux/tet hybrid promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934024 BBa_K934024]). which is important part for our band detect system. Plux/tet-GFP ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934025 BBa_K934025]) showed fluorescence only in the presence of both 3OC6HSL and aTc. In the presence of both inducers, the culture showed about 200-fold higher fluorescence intensity than that in the absence of both inducers. (Fig2-1-1-4).

Third, to represent “Scene3 Romeo’s suicide” and “Scene4 Juliet’s suicide”, we constructed two communication inverters: Plux-LacI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934026 BBa_K934026]) and Plas-LacI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934016 BBa_K934016]). Plux-LacI expresses LacI repressor in the presence of 3OC6HSL. On the other hand, Plas-LacI expresses LacI repressor in the presence of 3OC12HSL. Moreover, we conducted a simulation to confirm the feasibility of our cell-cell communication system

Fig2-1-1-1,[Go to
"Construction_of
the_3OC6HSL-dependent
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Fig2-1-1-2,[Go to
"Construction_of
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Fig2-1-1-3,[Go to
"Positive feed back assay"]

Fig2-1-1-4,[Go to
"Band detect system"]

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"

Fig2-1-2-1, the story of “Romeo and Juliet"

The story that we reproduce is divided into four scenes.(Fig2-1-2-1)

(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.

Fig2-1-2-2, Circuit design for “Romeo and Juliet”

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 E.coli play “Romeo and Juliet”. (Video1) The cell-cell communication system is composed of two types of engineered E.coli each of which plays Romeo and Juliet, respectively. We represented the four scenes with concentration of signal molecules 3OC6HSL and 3OC12HSL. 3OC6HSL is synthesized by LuxI enzyme in Romeo cell, 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, 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.

Third, to realize “Scene3 Romeo’s suicide” in response to Juliet’s deathlike sleep, we designed communication –inverter dependent suicide system in Romeo cell.

Finally, to realize “Scene4 Juliet’s suicide” in response to Romeo’s suicide, we also designed communication-inverter dependent suicide system in Juliet cell.

Fig2-1-2-3, Scene 1 Fall in love: positive feedback system

The love between Romeo and Juliet is realized by positive feedback of cell-cell communication signals (Fig2-1-2-3). 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.

Gene activations in the positive feedback proceed in the following manner. In Juliet cell, 3OC6HSL is bound by LuxR to form LuxR-3OC6HSL complex. The complex activates the expression of LasI. The accumulation of LasI, 3OC12HSL-production enzyme, increases concentration of the signaling molecule in the medium. In the mean time, LasR-3OC12HSL complex in the Romeo cell similarly activates the expression of LuxI, which produce 3OC6HSL. Thus the concentration of both signal molecules in the medium becomes higher than that in initial conditions. The higher concentration of signal molecules, the more complexes exist in each cell. Then the activation of LuxI and LasI gets stronger. As a result, the increase of the signal synthesis is accelerated.

Fig2-1-2-4, Scene 2 Juliet’s deathlike sleep: 3OC6HSL-dependent band detect system

The “Juliet’s deathlike sleep”, which is represented by the stop of 3OC12HSL production in a high concentration of Romeo signal 3OC6HSL, is realized by 3OC6HSL-dependent band detect system (Fig2-1-2-4). The band detect system is composed of Plux promoter, new Plux/tet hybrid promoter, and two regulator proteins. One of the proteins, LuxR, is expressed constitutively. The other one, TetR, is under Plux promoter, which is regulated by LuxR-3OC6HSL complex. The target gene of the band detect system is under Plux/tet hybrid promoter, which 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 though Juliet cell is alive.

Fig2-1-2-5, Scene3 Romeo’s suicide: communication –inverter dependent suicide system in Romeo cell

“Romeo’s suicide” in response to Juliet’s deathlike sleep is realized by communication-inverter dependent suicide system in Romeo cell (Fig2-1-2-5). In the presence of 3OC12HSL, LacI whose expression is regulated by LasR-3OC12HSL complex represses the expression of a lysis gene. Thus, Romeo cell keeps alive when Juliet cell produces 3OC12HSL. However, when Juliet cell is in death like sleep, supply of 3OC12HSL is stopped. Then the expression of LacI is also stopped in Romeo cell. In the absence of LacI, the lysis gene is expressed and Romeo cell dies.

Fig2-1-2-6, Scene4 Juliet’s suicide: communication-inverter dependent suicide system in Juliet cell

“Juliet’s suicide” in response to Romemo’s suicide is realized by communication-inverter dependent suicide system in Juliet cell (Fig2-1-2-6). Juliet cell can keep alive in the presence of 3OC6HSL, because the lysis gene is repressed by LacI whose expression is regulated by LuxR-3OC6HSL complex. After the death of Romeo cell, supply of 3OC6HSL is stopped, so 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 subsystems 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 Fig2-1-4-1. The Variables are described in Table2-1-4-1.

[Detailed descriptions for Modeling development]

Fig2-1-4-1,The equations used in the model

Table2-1-4-1, the variables

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. Fig2-1-4-2 shows the result of the simulation about time-dependent change of the concentrations of the two signals. We verified the 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.

Fig2-1-4-2, time-dependent change of the concentrations of the two signals. The blue line represents the concentration of Romeo’s signals in the culture and the red line represents Juliet’s.

We set the initial values of variables and the parameters as follows: [see more]

Fig2-1-4-3, time-dependent change of the concentrations of the two signals.

Fig2-1-4-4: the story of “Romeo and Juliet”

In the gray area of Fig2-1-4-3, the concentration of two signals increases. It looks as if Romeo and Juliet fall in love.

In the green area of Fig2-1-4-3, 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 Fig2-1-4-3, 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. This represents the suicide of Romeo. In the story, he thought Juliet was dead, and killed himself.

In the pink area of Fig2-1-4-3, 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. 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 Juliet cells under 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.

(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 (Fig2-1-4-6). Second, we prepared the separately cultured cells system (Fig2-1-4-7). In that system, Romeo cells and Juliet cells are cultured separately.

Fig2-1-4-5, the behavior of signal concentration in the complete cell-cell communication system

Fig2-1-4-6, the behavior of signal concentration in the constitutively signal producing system

Fig2-1-4-7, the behavior of signal concentration in the separately cultured cells system

In Fig2-1-4-5, the behavior of signal concentration in the complete cell-cell communication system is shown. As a comparison, in Fig2-1-4-6, the concentration of Romeo signals and Juliet signals increases slightly at first, but starts to decline before rising sufficiently. In Fig2-1-4-7, the concentration of Romeo signals decreases while Juliet signals increase. 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.

(2)Band detect system

Next, we confirmed the function of the Band detect system. Fig2-1-4-8 and Fig2-1-4-9 show the concentration change of the output signals responding to the concentration change of input signals.

Fig2-1-4-8, the concentration change of output Romeo signals responding to the concentration change of input Juliet signals

In Romeo cells, the production of Romeo signals increases monotonically with the increase of Juliet signals.(Fig2-1-4-8)

Fig2-1-4-9, the concentration change of output Juliet signals responding to the concentration change of input Romeo 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(Fig2-1-4-9). 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.

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

Fig2-1-4-10,
(a)the concentration of Juliet signals
(b)the concentration of lysis proteins in Romeo cells
(c)the number of Romeo cells

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 Fig2-1-4-10, when the concentration of Juliet signals is high (Fig2-1-4-10(a)), 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 (Fig2-1-4-10(b)). As a result, the population of Romeo cells increases (Fig2-1-4-10(c)). On the other hand, on the Line(2) in Fig2-1-4-10, when the concentration of Juliet signals is low (Fig2-1-4-10(a)), the expression of lysis proteins in Romeo cells is promoted (Fig2-1-4-10(b)). Thus, the population of Romeo cells decreases (Fig2-1-4-10(c)).

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.

Fig2-1-4-11 (a)the concentration of Romeo signals (b)the concentration of lysis proteins in Juliet cells (c)the number of Juliet cells

(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 Fig2-1-4-11, when the concentration of Romeo signals is high (Fig2-1-4-11(a)), the expression of LacI(proteins that inhibit lysis gene) is promoted strongly in Juliet cells. Thus, the expression of lysis proteins in Juliet cells is inhibited (Fig2-1-4-11(b)). As a result, the population of Juliet cells somewhat increases (Fig2-1-4-11(c)). On the other hand, on the Line(2) in Fig2-1-4-11, when the concentration of Romeo signals is low (Fig2-1-4-11(a)), the expression of lysis proteins in Juliet cells is promoted (Fig2-1-4-11(b)). Thus, the population of Juliet cells decreases (Fig2-1-4-11(c)).

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.

Possible Synbio research area by using our achievement

When we focus on the change in individual number of E. coli cell, we can consider our project “Cell-cell communication” as an experimental ecosystem by synthetic biology. ([2][3] )

We consider the behavior of activating mutual signal between cell R and cell J as the biological mutualism. By extension our project “cell-cell communication”, we are able to simulate ecosystem in culture. We note this as to the future prospects of our project.

Reference

1. Basu S, Gerchman Y, Collins CH, Arnold FH, & Weiss R (2005) A synthetic multicellular system for programmed pattern formation. Nature 434(7037):1130-1134.

2. You L, Cox RS, Weiss R, & Arnold FH (2004) Programmed population control by cell-cell communication and regulated killing. Nature 428:868-871.