Team:Chiba/Project

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Revision as of 16:28, 29 October 2008

Abstract

E.coli time manager

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.

As signaling molecules, we utilize molecules associated with Quorum sensing, a phenomenon that allows bacteria to communicate with each other. Although different quorum sensing species have slightly different signaling molecules, these molecules are not completely specific to their hosts and cross-species reactivity is observed (1),(2). Communication using non-endogenous molecules is less sensitive than the original, and requires a higher signal concentration to take effect. This reduced sensitivity results in the slower activation of receivers, thus creating a system in which different receivers are activated after different amounts of time following signaling molecule release.

Introduction

Fig.1 Project design

Many electronic devices we use in our daily lives have the ability to keep track of time. For example, a VCR is able to record a TV program at a pre-set time, and a microwave automatically stops heating after a set amount of time. automatically stop heating when the right time comes. Using these temporal pre-programming functions, we have been liberated from either staying up late to watch a European soccer game or from worrying about our popcorn being burned black while yelling and shouting to the match we have videotaped. In this way, the timer function has revolutionized our lifestyle.

We thought the same applies to the biotechnology; we would like to freely implement the 'timer switches” to various biological functions, preferably both independently and in parallel format. These “functions” include sensors, synthesizers, or degraders of bioactive compounds/ materials, transportation and secretion machineries, communications, getting/ sticking together, proliferation and cell death. If successful, we will be able to program exceedingly more complex complex behaviors in cellular systems.

As one of the thousands of possible applications, we are trying to construct a temporal imaging system using E. coli 'ink' that differ not in color but the 'timing' at which they are a certain color (fig). Over time, parts of images (or characters) are getting visible one by one, making animated message/ picture. If the coloration process proceeds to completion, the message is obscured. Only when the message is observed at the correct timing during the coloration process is any useful information obtained. Such a system should be useful for communication security: we can convey our message to only those that know the exact moment they should take a look. After a while, the message is gone and cannot be retrieved.

Project Details

Fig. System design

We designed the "switching consortium" that works like an water clock(Water clock-wikipedia.en). Here is how it works (Fig X). 1. Sender cells slowly generates the signal molecules AT A CONSTANT RATE. The signal molecules are non-degradable so that they get accumulated (linearly) over time. 2. Receivers detect the signals and then activate the genetic switch on. It happens only when the signal concentration reaches the switching threshold of the receivers. This way, the entire system behave like a delay switch. 3. Either by changing the receiver sensitivity or rate of signal accumulation, one can freely control the delay time of the switch. Using the switches with various time-of-delay, one can sequentially activate many different cellular functions

Signaling System

In this project, we use acylated homoserine lactones (AHLs), the signaling molecules used in quorum sensing system among gram negative bacteria.

Senders express LuxI or its family enzymes, the producer of AHLs. LuxIs are placed under the control of constitutive (Tet) promoter; each cell generates AHL more or less in a constant rate. AHL can freely permeate the cell membranes and get detected by neighboring cells.

Receivers constitutively express LuxR proteins (or its homolog), the detector of AHLs. When AHLs bind the LuxR proteins, AHL-LuxR complex activate the Lux promoter on. The switching threshold (in [AHL]) is determined by the affinity between AHL and LuxR. (文献)。 (more about quorum sensing)

System Design

Signal Molecule Sender Phase

Signal Molecule Receiver Phase

Chiba project design.jpg Signal Molecule Quencher

Experiments and Result

Signal Molecule Sender Phase

• （新）Signal Molecule Sender Phase

• （旧）more about Sender experiment and result

Signal Molecule Receiver Phase

English:

日本語:AHLを合成するSenderだけではなく、AHLを受け取る側のReceiverを変えれば、その応答時間を変えることができる。そこで私たちは、以下のいくつかの方法を考えた。

1. 一種類のSender（AHL<--LuxI）に対して、由来生物の異なるレシーバタンパク質でそれを受信する.
2. レシーバータンパク質であるLuxRに変異を入れることで、AHLに対する応答感度を上下させること.
3. レシーバーのコピーナンバーを変える.

Quorum-Sensing Crosstalk Fig.　 Crosstalk クオラムセンシングにおける、レシーバータンパクを変えてクロストークを起こさせる。 センダーを変えたときと同様に、他種生物由来のレシーバーでもAHLに応答することは知られている(1)(2)。 本来の組み合わせとは異なるAHLを受け取るレシーバーの応答時間は遅くなり、遺伝子発現が遅くなる。 more about Receiver phase crosstalk Fig.Time Delay Test LuxI-LuxR Fig.Time Delay Test LuxI-LasR Fig.Time Delay Test LuxI-RhlR more about experimental result

Plasmid Copynumber Fig. copynumber レシーバーのコピーナンバーを変えることで、応答までの時間を変える コピーナンバーを変えれば、レシーバーによるLuxRの合成量は変化する AHLを受け取るLuxRが変わるので応答閾値までの時間が変わるのだ more about Plasmid Copy number Fig. Time Delay Test クオラムセンシングに関わる遺伝子のベクタープラスミドのコピーナンバーを少なくすることで、遺伝子発現が遅くなる それと同時に、遺伝子発現の最大値自体も少なくなってしまう more about experimental result

LuxR mutant (Under construction)

レシーバータンパク質であるLuxRに変異を入れることで応答感度を上下させる^(3),(4)

Fig. LuxR mutant

Signal Molecule Quencher

Design

AHL reporter with aiiA Express LuxR and aiiA constantly. AiiA degrades AHL as signaling molecule. Express GFP when the AHL concentration exceed the capacity of aiiA. This enables the delay of the activation time of receiver. more about AiiA Receiver Phase

Fig.　 Time Delay Test more about AiiA Receiver Experiment

Demo Experiments

Demo ~Senders~

一番時差が見られたSender遺伝子のLuxIとLasIをつかってデモ実験を行った。

LuxIおよびLasIの遺伝子がそれぞれ組み込まれた大腸菌（XL10G）と、

LuxRの遺伝子が組み込まれた大腸菌（BW）を液体培養したものを 液量１：１で混ぜて、それらのGFPが発現するのを目視で観測した。

Results

Green region: sender=LuxI (100 uL), Red circular region: sender=Las I (100uL).
Receiver=?????? (???? uL)

0 h

4 h (Lux I GFP detected)

8 h (Lux I GFP and Las I GFP detected)

--toyota -->more about Demo experiments detail

Demo ~Receivers~

English:
日本語:
固体培地中にセンダー[http://partsregistry.org/Part:BBa_S03623 BBa_S03623],(Ptet-LuxI) を混ぜ、固体培地表面にレシーバーのコロニーをN.Cフィルターで移す。 センダーの作るAHLは培地中を移動し、表面のレシーバーがAHLを一定濃度感知すればGFPを発現 する。一種のセンダーに対し、様々な種類のレシーバーを用いることで時間差が生じることを確認する。

用いるレシーバーは・・・

・シグナル自体を分解するAiia を利用する

・レシーバーの遺伝子回路を含むプラスミドのコピーナンバーの変化

・レシーバータンパク質であるLuxRに変異を入れる 　　　　　

・確認の仕方
N.Cフィルターをはった個体培地を37°Cで培養し、時間(30min?)ごとにUVをあててGFPが見えるかチェックする。
香取

Results

--->more Demo experiments detail

Conclusion

シグナル分子を3OC6HSLから3OC12HSLに変更することで、LuxRの応答時間を二時間遅らせることができた.

--Yoshimi 11:09, 29 October 2008 (UTC)

Future Work

以下の実験を行うことで、Delayed Timeのバリエーションがもっと増えるだろう。

• Sender実験とReceiver実験で時間差を作り出すことが出来たものを組み合わせる。
• LuxRのミューテーション。
• ポジティブフィードバックループを利用する。

Applications

時間の蓄積（クロストーク）を使うと…

セキュリティーシステム GFPなどの目で確認できる文字（情報）を浮き上がらせてから、時間をおいて周りの菌にGFPを発現させる。
そうすとことによって、最初の文字（情報）を塗りつぶす。
もともと存在していた情報を消すことができる。

賞味期限装置 食品が傷む原因は細菌の活動が影響している。細菌の活動は温度に依存するので、温度変化を入力とする。
食品が傷む前にGFPを発現して私たちに知らせてくれる。
どれだけ時間変化が長くとも、短くとも私たちに知らせてくれる。

積分装置 消えてしまう入力（たとえばUV被ばく、汚染物質）をセンダーがAHLを出すことによって入力を記憶しておく。
AHLがたまり、危険量に達したことを知らせてくれる。
（杉山）

順番スイッチ 発現時間の異なるdelayed　switchを組み合わせることで、決められた順序が変わることなく順々にスイッチが入っていく自動的なシステムが作れる。

references

1. [http://www.pnas.org/content/100/suppl.2/14549.full Michiko E. Taga. Bonnie　L.Bassler.:Chemical communication among bacteria.PNAS.November 25, 2003,100.suppl.2]
2. [http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.micro.55.1.165?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dncbi.nlm.nih.gov Melissa B. Miller and Bonnie L. Bassler.:QUORUM SENSING IN BACTERIA.annurev.micro.55.1.165.2001]
3. [http://www3.interscience.wiley.com/journal/119124142/abstract M.K Winson et al.:Construction and analysis of luxCDABE-based plasmid sensors　for investigating N-acyl homoserine lactone-mediated quorum sensing.FEMS Microbiology Letters 163 (1998) 185-192]
4. [http://partsregistry.org/Part:BBa_F2620:Specificity BBa_F2620:Specificity]
5. [http://authors.library.caltech.edu/5553/ C. H. Collins.et al.:Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones.Mol.Microbiol.2005.55(3).712–723]
6. [http://mic.sgmjournals.org/cgi/content/abstract/151/11/3589 B. Koch. et al.:The LuxR receptor: the sites of interaction with quorum-sensing signals and inhibitors.Microbiology 151 (2005),3589-3602]

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