Team:Tokyo Tech/Project/Ninja State Switching

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Revision as of 11:51, 26 September 2013


Contents

1.Introduction

Through the human practice, we felt the importance of the explanation of the genetic programming in synthetic biology with interesting story. Thus we decided to make E. coli play "Ninja" story. In this story, there are three characters, so we prepared three E. coli which have different plasmids for each role, One is the hero, E. Ninja and the others are E. civilian and E. samurai (Fig1-1).

E. ninja switch between two states depending on whether there is E. civilian or E. samurai (Fig1-2). First, E. civilian is around E. ninja. E. civilian emit the small intercellular molecules 3OC6HSL. When E. ninja detect 3OC6HSL, E. ninja switch into the “mimic state” (Fig1-2 Step1). E. ninja maintain “mimic state” even after E. civilian go away (Fig1-2 Step2). Then the E. samurai come. E. samurai emit a different small intercellular molecules, 3OC12HSL. When E. ninja detect 3OC12HSL, E. ninja switch into the “attack state” (Fig1-2 Step3). E. ninja continue to maintain “attack state” even after the E. samurai leave (Fig1-2 Step4). Finally E. civilian return. E. ninja detect 3OC6HSL again, and switch back to the “mimic state” (Fig1-2 Step5).

To try to put this story into practice, we designed two genetic circuits: Signal-dependent state change circuit and Signal-dependent state change circuit with crosstalk circumvention (Fig 2-1). Both of the circuit has a toggle switch subcircuit and toggle-repressor overexpression subcircuit induced by intercellular communication molecules. In the case without cross talk, each of intercellular communication molecules, 3OC6HSL and 3OC12HSL, can switch the state of the toggle as reported by Collins group. (H. Kobayashi et al., 2004) However, crosstalk between 3OC12HSL-LasR complex and lux promoter disrupts our scenario. In order to circumvent the cross talk, we thus introduced two modifications. One is replacement of lux promoter for the overexpression with lux/tet hybrid promoter produced Tokyo tech 2012 (BBa_934024). The other is addition of a repressor network containing CI434 and TetR. We confirmed cross talk circumvention on the hybrid promoter experimentally (FigX) and also showed the circumvention in the whole network by mathematical modeling (FigX). The results correspond to the following scenario. When E. ninja receive 3OC6HSL, 3OC6HSL-LuxR complex activates Plux/tet hybrid promoter, and E. ninja switch into LacI dominant "mimic state." On the other hand, when E. ninja receive 3OC12HSL, 3OC12HSL-LasR complex activates las promoter, and E. ninja switch into CI dominant "attack state" (Fig2-1) because of crosstalk inhibition by tetR expressed in the LacI-dominant "mimic state."


2.Gene Circuit in Theory

To try to put this story into practice, we designed two genetic circuits: Signal-dependent state change circuit and Signal-dependent state change circuit with crosstalk circumvention\

2-1.Signal-dependent state change circuit

Originally, we designed the Signal-dependent state change circuit by combination with bistable "toggle switch"(T. Gardner et al., 2000) and toggle-repressor overexpression subcircuit induced by intercellular communication molecules. (Fig2-1). small molecules 3OC6HSL and 3OC12HSL. can switch the state of the toggle as expressed in the following step-by-step descriptions. The toggle switch has two promoter-gene sets Pλ and Plac. Pλ and Plac promoter are repressed by CI and LacI, and coding regions of these proteins CI and LacI are in downstream of each promoter gene (Fig2-1). With the combination of the tow subcircuits, our genetic circuit switches into two stable states by inducing of intercellular molecules, this change proceed step-by-step. Note that the explanation of below is in a situation without cross talk problem which we addressed in the latter part of this page.

Step1. When E. civilian emit 3OC6HSL and E. ninja receive these molecules, the 3OC6HSL-LuxR complex activate lux promoter. LuxR is expressed constitutively in E. ninja, so 3OC6HSL combine LuxR when 3OC6HSL is received by E. ninja. Then protein LacI start to be expressed because its upstream's lux promoter is activated by 3OC6HSL-LuxR complex, and LacI repress lacI promoter. Finally, E. ninja switch into the "mimic state" where LacI is expressed from lux promoter and Pλ on toggle switch (Fig2-2-1). (H. Kobayashi et al., 2004)

Step2. When E. civilian leave from around E. ninja, lux promoter activation disappears because 3OC6HSL-LuxR complex are not formed by lack of 3OC6HSL. However, LacI continue to be expressed from Pλ on toggle switch. Therefore E. ninja maintain the "mimic state" (Fig2-2-2).

Step3. Next, E. samurai come along. When E. samurai emit 3OC12HSL and E. ninja receive this molecules, the 3OC12HSL-LasR complex activate las promoter. LasR is expressed constitutively in E. ninja, so 3OC12HSL combine LasR when 3OC12HSL is received by E. ninja. Then protein CI start to be expressed because its upstream's las promoter is activated by 3OC12HSL-LasR complex, and CI repress Pλ. Finally, E. ninja switch into the "attack state" where CI is expressed from las promoter and lac promoter on toggle switch (Fig2-2-3).

Step4. When E. samurai leave from around E. ninja, las promoter activation disappears because 3OC12HSL-LasR complex are not formed by lack of 3OC12HSL. However, CI continue to be expressed from Plac on toggle switch. Therefore E. ninja maintain the "attack state" (Fig2-2-4).

Step5. When E. civilian return and in the same situation like spet1, E. ninja switch into "mimic state" again. When E. civilian emit 3OC6HSL and E. ninja receive these molecules, the 3OC6HSL-LuxR complex activate lux promoter. Then protein LacI start to be expressed because its upstream's lux promoter is activated by 3OC6HSL-LuxR complex, and LacI repress lacI promoter. Finally, E. ninja switch into the "mimic state" where LacI is expressed from lux promoter and Pλ on toggle switch (Fig2-2-5).

2-2.The problem: crosstalk

Though we described an ideal case in the above description, this genetic circuit had a crosstalk problem. In the case of this genetic circuit, LasR, which turn E. ninja into the "attack state," activates not only las promoter but also lux promoter. The crosstalk of LasR confuses E. ninja when E. samurai come. When E.ninja received intercellular molecules 3OC12HSL from E. samurai, 3OC12HSL-LasR complex activate las promoter and also activate lux promoter (Fig2-3-1).

The crosstalk between intercellular molecules 3OC6HSL and 3OC12HSL has been reported in a scientific paper. (Gray KM et al., 1994), We also quantified the crosstalk to confirm whether 3OC12HSL-LasR complex actually activate both las promoter and lux promoter (FigX). We prepared two different plasmids; one with GFP-gene regulated by las promoter (パーツ番号) and the other with GFP-gene regulated by lux promoter (パーツ番号). We introduced each plasmid into E. coli which have another plasmid to express LasR constitutively. Then, we added 3OC6HSL or 3OC12HSL in the culture growing these two strains. The result of this experiment is shown in Fig2-3-2. When we added 3OC6HSL, we could not confirmed adequate expression of GFP in both strains. This result shows little crosstalk between C6-HSL and LasR. On the other hand, when we added 3OC12HSL, we confirmed expression of GFP in both strains. This shows that 3OC12HSL-LasR complex activate not only las promoter but and lux promoter due to the crosstalk.

2-3.Signal-dependent state change circuit

with crosstalk circumvention

We thought of two possible approaches to solve the crosstalk. One is protein/promoter engineering and the other is gene network engineering. We then decided to choose gene network engineering to solve crosstalk problem.

We designed new genetic circuit shown shown in fig2-3-3. Our new gene circuit can circumvent crosstalk between intercellular molecules 3OC6HSL and 3OC12HSL (Fig2-3-3). Two new proteins, CI434 and TetR are added to the original Signal-dependent state change circuit. In addition, the lux promoter is changed to the Plux/tet hybrid promoter, which is Plux/tet hybrid promoter is repressed by TetR. In the case when changing from the "mimic state" to the "attack state," the tetR presence due to the absence of CI434 inhibits expression from the hybrid promoter. Without this inihibition, LasR protein, activated by 3OC12HSL from E. samurai, binding to luxR-binding sequence of the hybrid promoter would stimulate Lac I expression from the promoter. This expression, in addition to the CI expression from the las promoter to makes E. ninja confused. However, using new gene network, crosstalk is circumvented and E. ninja switch "mimic state" into "attack state" normally. This is because Plux/tet hybrid promoter is repressed by TetR (Fig2-3-4). Contraly, in the attack state, Plux/tet hybrid promoter is not repressed due to the absence of tetR. This is because expression of tetR is repressed by CI434. So when E. civilian come, Plux/tet hybrid promoter is activated by 3OC6HSL-luxI complex, then E. ninja switch into “mimic state.“

校正ここまで


3.crosstalk circumvention assay

Introduction

Our purpose is to check whether the hybrid promoter Plux/tet would be repressed or not when C12-LasR complex and protein TetR are activated.(Fig9) We tried to compare the amount of crosstalk in the presence or absence of the TetR inhibitor aTc. aTc weakens the bondings of TetR and TetO. Therefore, if the level of GFP expression of the cells we added C6 and aTc was higher than that of the cells we added only C6, it is proved that hybrid promoter Plux/tet is repressed by TetR working. Similarly, if the level of GFP expression of the cells we added C12 and aTc was higher than that of the cells we added only C12, it is proved that the crosstalk can be suppressed by TetR and the hybrid promoter.

Construction

We made a simple crosstalk circumvention system and named it “Crosstalk Circumvention Switch”. (Fig 10) To construct the circuit in above, we ligated Pcon-RBS-LasR-TT(K553003) and Plux/tet-RBS-GFP-TT(K934025) as the reporter plasmid. We used Pcon-RBS-LuxR-TT-Ptrc-RBS-TetR-TT as the regulator plasmid.

We prepared six conditions as follow. •A-1) Culture containing Crosstalk Circumvention Switch cell with 3OC6HSL induction •A-2) Culture containing Crosstalk Circumvention Switch cell with 3OC12HSL induction •A-3) Culture containing Crosstalk Circumvention Switch cell with DMSO ( no induction) •B-1) Culture containing Crosstalk Circumvention Switch cell with 3OC6HSL and aTc induction •B-2) Culture containing Crosstalk Circumvention Switch cell with 3OC12HSL and aTc induction •B-3) Culture containing Crosstalk Circumvention Switch cell with DMSO and aTc (no induction)

Result

In the graph below (Fig11), the level of GFP expression in cells where TetR is active is clearly lower than when TetR is inhibited. This fact could be confirmed in results about C12 and C6. In short, The graph below shows that Plux/tet is repressed by TetR precisely. Furthermore, the graph below shows that there is a great difference between GFP fluorescence intensity of C6+aTc and that of C12+aTc. In short, this shows that an affinity of LuxR-C6 complex toward Plux/tet is mightier than LasR-C12 complex.

Through this assay, we confirmed points below. ・Plux/tet is precisely repressed by TetR. This shows crosstalk circumvention. ・An affinity of LuxR-3OC6HSL complex toward Plux/tet is stronger than LasR-3OC12HSL complex.


4.


5. mathematical modeling

We made a mathematical model to forecast whether this solution might work in the complete circuit. When we made a mathematical model, we used results from assay. ・How affinities of LuxR-3OC6HSL complex and LasR-3OC12HSL complex toward Plux/tet are different. ・How affinities of LasR toward Plux and Plas are different. Our result is shown in two graphs below(Fig12, Fig13). Look at the orange line and the green line. These lines predict that both switches should work, enabling switching both from attack to mimic state, and vice-versa.


6. application

Our crosstalk circumvention systemの意義を書いたTopicに。拡張性かなOur crosstalk circumvention system gives you more flexibility to design genetic circuits. Our crosstalk circumvention system has a network topology composed of two repressor proteins and one repressor and one hybrid promoter. Along with the topology, one can just choose in any combination of sets of repressor protein and promoter. This system can be used for all genetic circuit that has other crosstalk.

Also the whole E. ninja system would be useful in industrial use. Intercellular molecules, 3OC6HSL and 3OC12HSL affect several cells simultaneously, and the number of cells can be controlled by the concentration of intercellular molecules. So our system is a switch that can control the ratio of ON and OFF. This system enables industrial production that always meets the demand.

Furthermore, the switch in E. Coli doesn’t need electricity, they grow explosively with just a small amount of LB. E. Coli’s exponentially growth gives us great energy efficiency,


7. Reference

1. Timothy S. Gardner (2000) Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339-342

2. Gray KM (1994) Interchangeability and specificity of components from the quorum-sensing regulatory systems of Vibrio fischeri and Pseudomonas aeruginosa. Journal of bacteriology 176(10): 3076–3080.

3. Hideki Kobayashi (2004) Programmable cells: Interfacing natural and engineered gene networks. vol. 101 no. 22 8414–8419