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 co-existed.(Fig9). C12-LasR-dependent activation of Plux promoter is known to be problem in synthetic biology and we confirmed this crosstalk activation (Fig). We then compared, for the first time, the amount of crosstalk for Plux/tet hybrid promoter in the presence or absence of the TetR inhibitor aTc. The bindings between TetR protein and TetO sequence on DNA is known to be weakened by aTc. Tokyo tech 2012 indeed showed that the GFP expression of the cells in which both of C6 and aTc were added was higher than that of the cells in which only C6 was added. Similarly, if the GFP expression of the cells where we added both of C12 and aTc was higher than that of the cells where we added only C12, it is proved that the crosstalk can be suppressed by TetR.

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.

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. This difference was referred in our mathematical modeling.

Discussion

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.

mathematical modeling

Modeling of Ninja circuit: Cross-talk circumvention

(detailed expression is here(link))

In order to clarify the parameter sensitivities and dynamic characteristics in the of the circumvention of cross-talk between LasR and LuxR, we modeled Ninja circuit by using ODEs. To know how strong the CrossTalk prevention circuit could be, furthermore, we compared with two gene circuits which with or without CrossTalk prevention circuit.

Firstly we consider the situation that when E.Civilian comes to cautious state. The following graph shows the switching from cautious state to civilian state which is influenced by C6 came from E.Civilian. And we can know when E.Civilian comes on the time of 300min, CI cautious state will switch to civilian state.

TetR increases after LacI

During the switching from the cautious state to the mimic state, absence of TetR allows activation of Pluxtet hybrid promoter. Repression of TetR production, by CI434, is indeed important in the circuit. During the mimic state, TetR accumulates to plateau level. This presence of TetR is important to prevent crosstalk by LasR activated by C12AHL.

Secondary, we consider the situation that when E.samurai comes to the mimic state. When switching occurs from cautious state to shuriken state, CI is expressed. Not that LacI expression form the Pluxtet hybrid promoter is prohibited, due to the presence of tetR, even in the presence of C12-LasR complex which can bind to the hybrid promoter for its activation. After C12 decomposition, shuriken state should switch back to cautious state.

Interestingly, CI expression oscillates and converge by C12AHL induction. This is because not only the toggle switch, but also there is a repressilator by combination among TetR, LacI and CI434. Note that there is difference in CI concentration between the shuriken state and the cautious state. This difference will be used for the decision making whether E.ninjya releases syuriken or not.

Then we compared the behavior of the E .Coli in the presence or absence of the crosstalk circumvention circuit. We succeed in confirming that the crosstalk circumvention circuit not only suppresses the crosstalk but also makes the switching faster.

The following graph shows the changing of LacI and CI. The solid line stand for the case with CrossTalk prevention circuit, and the dotted line stands for the case without CrossTalk prevention circuit. When there are certain amount of C12 production, LacI is produced in a certain amount in the toggle without CrossTalk prevention circuit, and in contrary, the Crosstalk would not be conspicuous. But in the other circuit which there is CrossTalk prevention circuit, LacI would not be produced and switching to the CI state is conspicuous.

For confirming the efficiency of those two toggle switches, we set LacI as the horizontal axis and CI as the vertical axis, then we plot the changing out. We can get the conclusion that the switching is much faster in the circuit with CrossTalk prevention circuit.

6. application

Our crosstalk circumvention system gives more flexibility to design genetic circuits because this system has a simple 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 various genetic circuits that has other crosstalk.

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