Team:Chiba/Project

The Project

1. Introduction
2. Project Design
3. Experiments, Results & Discussion
   1. Making LuxR Mutants
   2. Characterization
   3. For improving pictures
   4. Demonstration
4. Conclusion

E.coli Time Manager Since 2008

Introduction

It has been demanded in biological engineering that making the function that organisms sense the passage of time. We can utilize the excellent functions of organisms such as material synthesis or sensing as a device for drug delivery system or physical exam when these functions have been time-controlled as we like. However we can control cells individually, without method to control them all together, advantage of excellent functions may attenuate. Then we want to make the timer that cells works concurrently.

(手術予定)

①Timer創ります！

②E. coli Timer完成予想図

③Timerを創る方法は様々（diffusion of molecules, switching（oscillator, communication, arabinose, plac, etc... ））であるが、なぜ細胞通信を選んだのか。 そして、昨年は2段階の通信しか創れなかった。バリエーションが乏しい。

④作戦変更（Sender側をかえたCrosstalk通信にはバリエーションを創るのに限界があるので、今年はReceiver側の調節をする。）目指すは時計の数字分の、１２通りの時間差をつくりたい！

Project Design

For making some timers without need of synchronizer system and that can be used in a single system we try to make following design.

複数細胞の同時コントロール

2008 Melbourne, 2007/2008 Paris, and 2008 NYMU dealed in a bacterial timer but in these project it needs the synchronizing system to make the behavior of cell population synchronized. Then we have aimed at a making of the function that can control a mass of cells all together without synchronizing system. We have used cell-cell signaling system for this. By using it, massive numbers of cells in the system can share the time they fell.

Signaling System

In this project, we use acylated homoserine lactones (AHLs), signaling molecules used for [http://en.wikipedia.org/wiki/Quorum_sensing quorum sensing] in gram negative bacteria. Senders express LuxI or similar enzymes, which catalyze the production of AHLs, under the control of a constitutive (Tet) promoter. Each cell thus generates AHL more or less at a constant rate. AHL can freely permeate cell membranes and are detected by neighboring cells. Receivers constitutively express LuxR proteins (or a similar ortholog), the protein that detects AHL concentrations. When AHLs bind LuxR proteins, the AHL-LuxR complex activates the Lux promoter. The threshold [AHL] at which switching occurs is determined by the affinity of AHL for the particulr LuxR ortholog. about quorum sensing)

Constructing A Delay Switch (Since 2008)

We are constructing delay switches to control/preset the timing of target gene expression. Our project uses two classes of bacteria: senders and receivers. Senders produce signaling molecules, and receivers are activated only after a particular concentration of this molecule is reached. The combinatorial use of senders/receivers allows us to make a‘switching consortium’which activates different genes at the preset times.

Constructing A Delay Switch, Multiple Ways

In principle, there are three ways to delay the activation of chemical communications;

1. Silencing the Speakers: Rate of signal accumulation down-regulated, for instance, by slowing down the signal generators.
2. Desensitize Receivers: Switching threshold elevated, for instance, by using insensitive receiver/ reporter systems.
3. Partial Blocking: Decreasing the by chewing the signal up.

Inter-species communications!

We decided to go for the strategy inspired by Japanese-classic experience; Whenever we speak to somebody in English, we often experience a certain delay in activating the communication. We though this is exactly what we pursued in. See Exp #4. Same applies to the reverse, too. When somebody speaks to us, we definitely need some time (sometime infinite) to get activated. This is all in spite that he/ she was loud and clear enough. The less affinity (perception) we have to English, the longer we need to activate them.See exp #5.

-> Get more information about a Delay Switch!

Constructing A Flow of Activation

Conclusions