Assays for Positive feedback system

Fig2-1-3-1-1, Positive feedback system in the cell-cell communication

Introduction of the positive feedback

The positive feedback in the Romeo and Juliet cell-cell communication system is composed of two types of the engineered cells, 3OC6HSL-dependent 3OC12HSL producer cell (Plux-LasI cell) and 3OC12HSL-dependent 3OC6HSL producer cell (Plas-LuxI cell). In this positive feedback system, 3OC6HSL produced by Plas-LuxI cell activates the production of 3OC12HSL by Plux-LasI cell, and vice versa. For the implementation of the positive feedback system, several new Biobrick parts are required (Fig2-1-3-1-1).

Construction of the 3OC6HSL-dependent 3OC12HSL production module

For construction of the 3OC6HSL-dependent 3OC12HSL production module, we firstly constructed a new part Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]). Plux-LasI cell is an engineered E.coli that contains a 3OC6HSL-dependent LasI generator and a constitutive LuxR generator. As a 3OC6HSL-dependent LasI generator, we constructed a new Biobrick part Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]) by combining Plux promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_R0062 BBa_R0062]) and LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K081016 BBa_K081016] ). As a constitutive LuxR generator, we used Ptet-LuxR ([http://partsregistry.org/wiki/index.php?title=Part:BBa_S03119 BBa_S03119]). By introducing Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022] ) and Ptet-LuxR ([http://partsregistry.org/wiki/index.php?title=Part:BBa_S03119 BBa_S03119]) into E.coli strain JM2.300, we constructed Plux-LasI cell. Then we performed a reporter assay by using Las reporter cell to characterize the function of Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]) . As the negative control of 3OC12HSL production, we prepared 3OC12HSL non-producer cell (ΔP-LasI cell) that contains, in addition to Ptet-LuxR ([http://partsregistry.org/wiki/index.php?title=Part:BBa_S03119 BBa_S03119]), promoterless-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K081016 BBa_K081016]) instead of Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]) (Fig2-1-3-1-2).

Fig2-1-3-1-2, Las sender strain and Las reporter strain

The ⊿P-LasI cell does not produce 3OC12HSL even though 3OC6HSL exist. The supernatants of the cultures of these modules were used as the inducer in the reporter assay (Fig2-1-3-1-3).

Fig2-1-3-1-3, How to perform 3OC6HSL-dependent 3OC12HSL production assay

We prepared four conditions as follow.

A) Culture containing Plux-LasI cell without 3OC6HSL induction

B) Culture containing Plux-LasI cell with 3OC6HSL induction

C) Culture containing ⊿P-LasI cell without 3OC6HSL induction

D) Culture containing ⊿P-LasI cell with 3OC6HSL induction

Using the supernatant of the four culture conditions, we performed the reporter assay.

In the reporter assay, we used a Las reporter strain that contains Ptrc-LasR and Plas-GFP ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K649001 BBa_K649001]). Also, a reporter cell that expresses GFP constitutively and a reporter cell that does not express GFP were used as the positive control and the negative control, respectively.

Fig2-1-3-1-4, 3OC6HSL-dependent 3OC12HSL production

Fig2-1-3-1-4 shows fluorescence intensities by the reporter cells dependent on different conditions. Only when the supernatant of condition B was used, the fluorescence intensity of the Las reporter cell increased, while the supernatants of other three conditions did not affect. Comparing the results of the condition A and B, it can be said that with the induction of 3OC6HSL to Plux-LasI cell, the fluorescence intensity of the Las reporter cell increased by 20-folds. This result indicates that Plux-LasI cell produced 3OC12HSL in response to 3OC6HSL induction by the function of Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022] ). From this experiment, we confirmed that a new part Plux-LasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022] ) synthesized enough concentration of 3OC12HSL to induce the Las reporter cell. [Materials and Methods]

Construction of the 3OC12HSL-dependent 3OC6HSL production module

For construction of the 3OC12HSL-dependent 3OC6HSL production module, we firstly constructed a new part Plas-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934012 BBa_K934012] ). Plas-LuxI cell is an engineered E.coli that contains a 3OC12HSL-dependent LuxI generator and a constitutive LasR generator. As the 3OC12HSL-dependent LuxI generator, we constructed a new Biobrick part Plas-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934012 BBa_K934012] )by combining Plas promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K649000 BBa_K649000] ). and LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K081008 BBa_K081008] ). As a constitutive LasR generator, we used Ptrc-LasR. By introducing Plas-LuxI and Ptrc-LasR into E.coli strain JM 2.300, we constructed Plas-LuxI cell. Then we performed a reporter assay by using Lux reporter cell to characterize the function of Plas-LuxI. As the negative control of 3OC6HSL production, we prepared 3OC6HSL non-producer cell (ΔP-LuxI cell) that contains, in addition to Ptrc-LasR, promoterless-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K081008 BBa_K081008]) instead of Plas-LuxI (Fig2-1-3-1-5).

Fig2-1-3-1-5, Lux sender strain and Lux reporter strain

The ΔP-LuxI cell does not produce 3OC6HSL even though 3OC12HSL exist. The supernatants of the cultures of these modules were used as the inducer in the reporter assay (Fig2-1-3-1-6).

Fig2-1-3-1-6, How to perform 3OC12HSL-dependent 3OC6HSL production assay

We prepared four conditions as follow.

E) Culture containing Plas-LuxI cell without 3OC12HSL induction

F) Culture containing Plas-LuxI cell with 3OC12HSL induction

G) Culture containing ⊿P-LuxI cell without 3OC12HSL induction

H) Culture containing ⊿P-LuxI cell with 3OC12HSL induction

Using the supernatant of the four culture conditions, we performed the reporter assay. In the reporter assay, we used a Lux reporter strain that contains Ptet-LuxR and Plux-GFP ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K395100 BBa_K395100]). Also, a reporter cell that expresses GFP constitutively and a reporter cell that does not express GFP were used as the positive control and the negative control, respectively.

Fig2-1-3-1-7, 3OC12HSL-dependent 3OC6HSL production

Fig2-1-3-1-7 shows fluorescence intensities by the reporter cells dependent on different conditions. Only when the supernatant of condition F was used, the fluorescence intensity of the Lux reporter cell increased while the supernatants of other three conditions did not affect. Comparing the results of the condition E and F, it can be said that with the induction of 3OC12HSL to Plas-LuxI, the fluorescence intensity of the Lux reporter cell increased by 112-folds. This result indicates that Plas-LuxI cell produced 3OC6HSL in response to 3OC12HSL induction by the function of Plas-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934012 BBa_K934012]) From this experiment, we confirmed that a new part Plas-LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934012 BBa_K934012]) synthesized enough concentration of 3OC6HSL to induce the Lux reporter cell. [Materials and Methods]

Positive feedback assay ~Co-culture assay~

Fig2-1-3-1-1, Positive feedback system in the cell-cell communication

To accomplish complete positive feedback system, we mixed and co-cultured Plux-LasI cell and Plas-LuxI cell (Fig2-1-3-1-1). As a control co-culture, ⊿P-LasI cell and ⊿P-LuxI cell were mixed. For a trigger of the positive feedback system, we added the initial dose of 3OC6HSL (5nM) or 3OC12HSL (2.5nM) to the co-cultures. Including no-induction controls, we thus prepared six conditions (Fig2-1-3-1-8). To confirm that both 3OC6HSL and 3OC12HSL are produced in the positive feedback, the signals content in the supernatants of the co-cultures were evaluated by Las reporter strain and Lux reporter strain (Fig2-1-3-1-9).

Fig2-1-3-1-8, Six conditions prepared for positive feedback assay

Fig2-1-3-1-9, How to evaluate the positive feedback

In these co-cultures, following behavior would be expected. In the condition I, 3OC6HSL induces 3OC12HSL production of Plux-LasI cell. Then, 3OC12HSL synthesized by Plux-LasI cell induces Plas-LuxI cell. The concentration of 3OC6HSL thus increases compared to the initial amount. Increased amount of 3OC6HSL induces higher production of 3OC12HSL. As a result, the production of both signals increased in the condition I. In this situation, the concentration of 3OC6HSL is higher than the initial concentration of 3OC6HSL (ideal positive feedback behavior). Similarly in the condition II, total amount of 3OC12HSL also increases compared to the initial amount. In the conditions III and VI, no signal is produced because of the lack of inducer. In conditions IV and V, little amount of 3OC6HSL and 3OC12HSL are remained without degradation, respectively.

Fig2-1-3-1-10, Positive feedback assay

Fig2-1-3-1-10 shows that the fluorescence intensity of the Lux reporter cell in the condition I was higher than that in the condition IV. Similarly, the fluorescence intensity of the Las reporter in the condition II was higher than that in the condition V. From these results and circuit design described above, it is suggested that the appearance of positive feedback where cooperation of Plas-LuxI cell and Plux-LasI cell increase the production of 3OC6HSL and 3OC12HSL. [Materials and Methods]

Positive feedback assay~Time-dependent change assay~

We confirmed the appearance of the positive feedback from the co-culture assay above. To further confirm the positive feedback system, we characterized the time-dependent change of this positive feedback in the cell-cell communication. To observe time-dependent change of the positive feedback, we prepared four conditions of co-culture : Plux-LasI cell and Plas-LuxI cell coexisted in condition i, ΔP-LasI cell and Plas-LuxI cell in condition ii, Plux-LasI cell and ΔP-LuxI cell in condition iii, and ΔP-LasI cell and ΔP-LuxI cell in condition iv. For a trigger of the positive feedback system, we added the initial dose of 3OC6HSL (5 nM) to each co-culture.

Fig2-1-3-1-11,Four conditions prepared for Time-dependent change assay

To estimate the concentration of 3OC6HSL and 3OC12HSL produced by the positive feedback, the signals content in the supernatants of the co-cultures were collected at the time of 0, 0.5, 1, 2, 4 hours. The concentration of 3OC6HSL and 3OC12HSL produced by the positive feedback was evaluated by Las reporter strain and Lux reporter strain (Fig2-1-3-1-11). Fig2-1-3-1-11 also shows ideal signal production of the positive feedback in the cell-cell communication.

Fig2-1-3-1-12,Time-dependent change assay

Fig2-1-3-1-12 shows the characterization of time-dependent signal increase in the positive feedback in the cell-cell communication. Solid lines represent the fluorescence intensities of Las reporters in the four conditions. Dotted lines represent the fluorescence intensities of Lux reporters in the four conditions.

As compared red solid line with blue dotted line in the condition i (both Plux-LasI cell and Plas-LuxI cell coexist), the fig shows that the fluorescence intensity of Las reporter increases at first (0-1h), and then that of Lux reporter starts to increase (1-2h). This result indicates that the 3OC12HSL production in Plux-LasI cell was activated by initially added 3OC6HSL, whereas the 3OC6HSL production in Plas-LuxI cell was not activated till 3OC12HSL production in Plux-LasI cell reached sufficient level. This behavior strongly suggests the appearance of the positive feedback.

Fig2-1-3-1-12 also suggests that the advantage in signal production in the presence of both signal producers, Plux-LasI cell and Plas-LuxI cell. Only when Plux-LasI cell and Plas-LuxI cell were cultured together and allowed to communicate with one another, the fluorescence intensity of Las reporter and Lux reporter increased drastically. On the other hand, when either Plux-LasI cell or Plas-LuxI cell existed (condition ii and iii), drastic rearing in the GFP expression was not observed. Especially in the condition iii, GFP in the las reporter cell is expressed to some extent (about 2-folds lower than in condition i). This behavior shows that the production of 3OC12HSL in the condition iii cannot exceed the production of 3OC12HSL in the positive feedback state. This is because ΔP-LuxI cell cannot produce any 3OC6HSL to activate Plux-LasI cell in this condition, though initially added 3OC6HSL activated Plux-LasI cell and Plux-LasI cell produced 3OC12HSL to some extent. Moreover, it goes without saying that, the fluorescence intensity of both Las reporter and Lux reporter remained low level through observation in the absence of both signal producer cell.

In addition, low concentration of 3OC6HSL was detected by the Lux reporter in the condition ii, iii & iv, in which 3OC6HSL is not produced. However, fluorescence intensity in these conditions decreased with time. Therefore, it is indicated that these GFP expressions were derived from initially added 3OC6HSL.

From these overall results, we further confirmed the positive feedback in the cell-cell communication and characterized time-dependent change of the positive feedback. Only when the two types of signal producer cells were cultured together and allowed to communicate with one another, signal content in the supernatant of the co-culture drastically increased as compared with other conditions.

[Materials and Methods]

Conclusion of positive feedback system

In this study, we designed and implemented a positive feedback system in the cell-cell communication that is composed of the Plux-LasI cell and the Plas-LuxI cell.

First, we confirmed that the Plux-LasI cell synthesized enough concentration of 3OC12HSL to induce the Las reporter cell and the Plas-LuxI cell synthesized enough concentration of 3OC6HSL to induce the Lux reporter cell. In the process of the implementation, we constructed 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]), the new Biobrick parts that can be regulated by the induction of 3OC6HSL and 3OC12HSL, respectively.

By co-culturing of these two types of E.coli, we then confirmed that higher concentration of a signal than initial conditions was detected through production of the other signal. These results indicated appearance of the positive feedback.

To further confirm the positive feedback system, we also characterized the time-dependent change of this positive feedback in the cell-cell communication. The result indicates that the 3OC12HSL production in Plux-LasI cell was activated by initially added 3OC6HSL, whereas the 3OC6HSL production in Plas-LuxI cell was not activated till 3OC12HSL production in Plux-LasI cell reached sufficient level. This behavior strongly verifies the appearance of the positive feedback.

Only when the two types of E.coli were cultured together and allowed to communicate with one another, signal content in the supernatant of the co-culture drastically increase as compared to other conditions.

Band detect system

Introduction of band detect system

the original band detect system

To achieve our goals, we need the 3OC6HSL-dependent band detect system.

Fig2-1-3-2-1,
(a)the band detect system constructed by Ron Weiss Lab.
(b)Our new band detect system

In 2005, the first band detect system in synthetic biology was constructed by Ron Weiss Lab.([1] ) In that system (Fig2-1-3-2-1(a)), 3OC6HSL-LuxR complex activates the expression of LacIM1 repressor and CI repressor. Furthermore, CI repressor represses the expression of LacI. Then, both LacIM1 repressor and LacI repressor repress the expression of GFP. When the concentration of 3OC6HSL is high, the expression of LacIM1 is promoted strongly. As a result, the expression of GFP is repressed. When the concentration of 3OC6HSL is moderate, it results in the moderate level of CI and LacIM1. Because the repression efficiency of CI is sufficiently high, the LacI expression remains repressed. However, in this situation, the concentration of LacIM1, whose repression efficiency is low, is not sufficient to repress the GFP expression. Thus, GFP is expressed. When the concentration of 3OC6HSL is low, the expression of CI is only at low level. This enables the expression of LacI, then the expression of GFP is repressed again. In this way, the expression of GFP is activated by the band detect system under the particular range of the concentration of 3OC6HSL.

In this project, we invented the new band detect system (Fig2-1-3-2-1(b)). Compared with the old one, our new band detect system has a merit, that is constructed with fewer components. By reducing components in cells, we can avoid the useless complexity and the inhibition of cell growth. We employed the lux/tet hybrid promoter in the system. In the following, we described the details of our system.

our new band detect system

The band detect system for cell-cell communication system is composed of lux/tet hybrid promoter,Plux-TetR,and Pon-LuxR. In contrast to the Weiss lab’s band detect system, which take advantage of difference in activity between wild-type repressor and its mutant, we used a hybrid promoter for the system. The expression of target gene, LasI, is regulated by both of LuxR-3OC6HSL complex and TetR through binding of them to lux/tet promoter. The expression of TetR from lux promoter is also regulated by LuxR-3OC6HSL (Fig2-1-3-2-2). Thus, concentration of 3OC6HSL affects both of lux/tet hybrid promoter and lux promoter since Pon-LuxR constitutively express LuxR.

Fig2-1-3-2-2

When the concentration of 3OC6HSL is initial level, LuxR-3OC6HSL complex activates the expression of LasI. Though TetR is also expressed, its concentration is not enough to repress the expression of LasI.(Fig2-1-3-2-3)

Fig2-1-3-2-3

As the concentration of 3OC6HSL increases to moderate level gradually by the positive feedback system, the expression of TetR and LasI also increases. In this situation, the concentration of TetR is still not enough to repress the expression of LasI.(Fig2-1-3-2-4)

Fig2-1-3-2-4

When the concentration of 3OC6HSL is high level, the concentration of TetR reaches to the enough level to repress LasI expression(Fig2-1-3-2-5). For the implementation of the band detect system, lux/tet hybrid promoter is required.

Fig2-1-3-2-5

The repression of production of 3OC12HSL results in arrest of the 3OC6HSL supply from Romeo cell. Then the LuxR-3OC6HSL complex cannot form and the expression of LasI is stopped.(Fig2-1-3-2-6)

Fig2-1-3-2-6

Result

Fig2-1-3-2-7,lux/tet hybrid promoter assay

For construction of the band detect system, we developed a new part lux/tet hybrid promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934024 BBa_K934024]). The lux/tet hybrid promoter, which is composed of a LuxR operator and two TetR operators, activates the expression of the downstream gene only when LuxR-3OC6HSL complex exists and active TetR does not exist. To characterize the function of the lux/tet hybrid promoter, we constructed a part, Plux/tet-GFP ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934025 BBa_K934025]) by inserting the promoter in front of a GFP coding sequence. By using the reporter cell that contains Plux/tet-GFP and constitutive LuxR and TetR generator (PlacIq-LuxR-Ptrc-TetR), we measured the fluorescence intensity of the reporter cell. In the measurement, we confirmed the GFP expression under the four different combinations of two inducers, 3OC6HSL and aTc (anhydrous tetracycline). In the absence of the both inducers, the culture with lux-tet hybrid promoter-gfp showed the background–fluorescence intensity generated by promoterless-rbs-gfp on pSB3K3. The presence of either 3OC6HSL or aTc alone had little effect on increasing the fluorescence intensity. In the presence of both inducers, the culture showed about 500-fold higher fluorescence intensity than that in the absence of both inducers(Fig2-1-3-2-7). This result confirmed that the assembly of the LuxR operator and the two TetR operators integrated the inputs of 3OCH6HSL and aTc into the output of GFP transcription. [Materials and Methods]

Discussion

Fig2-1-3-2-8

Fig2-1-3-2-9

In this study, we improved lux/tet hybrid promoter parts by developing a new lux/tet hybrid promoter that is regulated by LuxR-3OC6HSL and TetR. Even though a former team had reported a lux/tet hybrid promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K176078 BBa_K176078]), their assay did not contain the inducer combination:3OC6HSL(-) and aTc(+).Thus, data of lux/tet hybrid promoter in Biobrick Registry was not sufficient(Fig2-1-3-2-9). In contrast, all four combinations were confirmed in our new Plux/tet-GFP ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934025 BBa_K934025]) Therefore, our work is considered as the improvement of the lux/tet hybrid promoter.

communication-inverter dependent suicide system

Introduction:

In order to reproduce the suicides of Romeo & Juliet’s, the communication dependent inverter system that express lysis protein only when the signals do not exist is required.To realize the communication dependent inverter system, we constructed Plas-LacI([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934016 BBa_K934016]) and Plux-LacI([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934026 BBa_K934026]) that express LacI to repress the target gene only when LasR-3OC12HSL and LuxR-3OC6HSL exist respectively.

3OC12HSL dependent

construction

We constructed a 3OC12HSL-dependent LacI generator ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934016 BBa_K934016]) by ligating PlasI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K649000 BBa_K649000]) to the upstream of rbs-LacI-ter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I732820 BBa_I732820]).

3OC6HSL dependent

construction

We constructed a 3OC6HSL-dependent LacI generator ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934026 BBa_K934026]) by ligating Plux ([http://partsregistry.org/wiki/index.php?title=Part:BBa_R0062 BBa_R0062]) to the upstream of rbs-LacI-ter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I732820 BBa_I732820]).

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 yellow 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 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.

a

Fig2-1-4-12, the behavior of signal concentration in the complete cell-cell communication system

Fig2-1-4-13, the behavior of signal concentration in the complete cell-cell communication system

Fig2-1-4-14, the behavior of signal concentration in the complete cell-cell communication system

Fig2-1-4-15, the behavior of signal concentration in the complete cell-cell communication system

Table3, the behavior of signal concentration in the complete cell-cell communication system