Team:Chiba/Project

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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　Burnt popcorn. He could have prevent this if he properly preset the timer...

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.

Fig. 2　Temporal imaging system. You can display secret message only for a short period of time. (reload to replay)

As one of the thousands of possible applications, we are trying to construct a metamorphosizing image using E. coli 'ink' that differ not in color but the 'timing' at which they are a certain color (fig2). 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 Design

Fig. 3　System design

We designed a switching consortium that works like a water clock (Water clock-wikipedia.en). Here is how it works (Fig 3).

1. Sender cells slowly generate signal molecules at a constant rate. The signal molecules are non-degradable (or virtually so in a reasonable time scale) so they get accumulated (linearly) over time.

2. Receivers detect the signal molecules and then activate the genetic switch to the on state, but only when the signal concentration reaches the switching threshold of a receiver. Receivers are activated by different signaling molecules at different rates. In this way, the entire system behaves like a delay switch sequence.

3. Either by changing the receiver sensitivity or rate of signal accumulation, one can freely control the delay length of the individual switches. Using switches with various times-of-delay, one can sequentially activate many different cellular functions.

Signaling System

In this project, we use acylated homoserine lactones (AHLs), signaling molecules used for 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.^(3),(4). (more about quorum sensing)

Constructing A Delay Switch, Multiple Ways

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

Silencing the Speakers: Rate of signal accumulation down-regulated, for instance, by slowing down the signal generators.
Desensitize Receivers: Switching threshold elevated, for instance, by using insensitive receiver/ reporter systems.
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.

List of Experiments

For details, click each of the index titles


Exp #1 Partial quenching of signals (jamming)	Exp #2 Balancing Player	Exp #3 Lux Mutants	Exp #4 Spoken to by Foreigners	Exp #5 Speaking to Foreigners

Others
- Searching for Reporters
- Demonstrations

Our (incomplete) metamorphosizing image

Fig.4a Flower should have blossomed!!!
Leaves, a stem and a flower was drawned with BBa_T9002, T9002-p15A, and AiiA Receiver, respectively on the plate containing BBa_S03623 (AHL sender)--->more about Temporal imaging system Demo experiments detail

Conclusion

In conclusion, we tried (and are trying) to device a series of delay-switches by designing the "switching consortia". We got limited, by certain success in generating delay switches.

We confirmed that various "foreign" AHL sender can activate the LuxR/ LuxP switch.
Using the 3OC12HSL (Las-type) instead of 3OC6HSL (Lux-type) as signaling input, we could delay the activation of the LuxR/ LuxP switch for 2 hours. As of today, Oct 29th), this is the slowest delay switch in our hand.
Interestingly, we kept failing to observe the crosstalk between Lux-sender and receivers from other organisms. Interestingly, we never seen the function of cinI/ cinR from Rhizobium leguminosarum. We have no idea why we could not reconstruct the communication (by this natural pairs).
Several other strategies have been tested, too. We have positive expectation especially on the engineering of LuxR protein.
Fiinally, we have provided 13 biobricks that will be useful in various future projects using cell-cell communications.

One of the technical challenge of our project was that the reporter genes are quite slow by itself to develop readable signal. There are significant time-lag between transcription initiation and the time the reporter start emitting the signal. We have screened many different reporters including lucifeases, lacZ, and other fluorescent proteins. With our experimental setup, they all looked more or less the same level (some were in the range of 30-60 mins in microscope, but all needed 90-120 mins to get visible by eye). For more precise PoPS analysis, we should have conduct blotting analysis, instead of fluorescent analysis.

Biological timer was previously designed by Missouri Miners (2007). In their system, they feed arabinose (input signal) to the bacteria, which consume the signal molecule. Upon eating up the molecule, the bacteria will generate GFP, signaling that time is up. This system is clever in that one can freely set the timer by adjusting the amount of arabinose fed to the cells. Our approach is more to make a series of timers. Obvious drawback of this approach is that we have to make new timer for each and every applications. Good thing about our system is that once created, we can run several timers simultaneously in a single pot; Combinatorial use of biological timers with different delay time would allows us to sequential switching of various genes, enabling more sophisticated control of the biological processes.

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 __NOTOC__
 ==Abstract==
-[[Image:Project design chiba QS.gif|frame|right|'''Fig.1''' Project desgin]]
+====　''E. coli'' time manager====
-::'''"Team : Chiba - 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.
-　　We control the timing of gene expression by using multiple signaling devices.To this end,we utilize molecules associated with Quorum sensing, a phenomenon that allows bacteria to communicate with each other.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.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 [http://www3.interscience.wiley.com/journal/119124142/abstract (1)],[http://partsregistry.org/Part:BBa_F2620:Specificity (2)]. Communication using non-endogenous molecules is less sensitive, and requires a higher signal concentration to take effect.This results in slower activation of receivers.
+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 [[Team:Chiba/Project#References|<sup>(1),(2)</sup>]]. 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==
-[[‎Image:Chiba-logo.gif‎||frame|'''Fig.2''' Team logo]]
+[[Image:Chiba_popcorn.jpg|thumb|right|'''Fig. 1　Burnt popcorn'''. He could have prevent this if he properly preset the timer...]]
+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 [[Team:Chiba/Project/Applications|complex behaviors]] in cellular systems.
+[[Image:Chiba_lifestinks.gif|frame|left|'''Fig. 2　Temporal imaging system.''' You can display secret message only for a short period of time. (reload to replay)]]As one of the thousands of possible applications, we are trying to construct a '''metamorphosizing image''' using ''E. coli'' 'ink' that differ not in color but the 'timing' at which they are a certain color (fig2). 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 Design==
+[[Image:Chiba fig3 2.png|right|frame|'''Fig. 3　System design''']]
+We designed a '''switching consortium''' that works like a water clock
+([http://en.wikipedia.org/wiki/Water_clock Water clock-wikipedia.en]). Here is how it works (Fig 3).
+. Sender cells slowly generate signal molecules at a constant rate.
+The signal molecules are non-degradable (or virtually so in a reasonable time scale) so they get accumulated
+(linearly) over time.
+. Receivers detect the signal molecules and then activate the genetic
+switch to the on state, but only when the signal concentration reaches
+the switching threshold of a receiver.
+Receivers are activated by different signaling molecules at different
+rates. In this way, the entire system behaves like a delay switch
+sequence.
+. Either by changing the receiver sensitivity or rate of signal
+accumulation, one can freely control the delay length of the
+individual switches. Using switches with various times-of-delay, one
+can sequentially activate many different cellular functions.
+===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.[[Team:Chiba/Project#References|<sup>(3),(4)</sup>]].
+[[Team:Chiba/about_qs|(more about quorum sensing)]]
+===Constructing A Delay Switch, Multiple Ways===
+In principle, there are three ways to delay the activation of chemical communications;
+#'''Silencing the Speakers''': Rate of signal accumulation down-regulated, for instance, by slowing down the signal generators.
+#'''Desensitize Receivers''': Switching threshold elevated, for instance, by using insensitive receiver/ reporter systems.
+#'''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 [[Team:Chiba/Project#List of Experiments|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.[[Team:Chiba/Project#List of Experiments|See exp #5.]]
-<br>
+==List of Experiments==
+===='''For details, click each of the index titles'''====
-==Our project==
+<center>
-[[Image:Image time manager chiba.jpg|frame|right|'''Fig.''' Our system]]
+{| style="border:0px;" cellpadding="0px" |
-従来:消えていく物質の存在時間によって時間調節する。Missouri Miners 2007 "A biological timer"
+|-
+| valign="center" align="center" width="20%"|
+[[Image:Chiba Igem 1.png|130px|'''Fig.4''' Chiba project design.jpg]]
+| valign="center" align="center" width="20%"|
+[[Image:Chiba Igem 2.png|172px|'''Fig.5''' ]]
+| valign="center" align="center" width="20%"|
+[[Image:Chiba Igem 3.png|172px|'''Fig.6''' LuxR mutant]]
+| valign="center" align="center" width="20%"|
+[[Image:Chiba Igem 4.png|130px|'''Fig.7''' ]]
+| valign="center" align="center" width="20%"|
+[[Image:Chiba Igem 5.png|130px|'''Fig.8''' ]]
-千葉:常に物質が一定の割合で蓄積されるとき、ある閾値超えるまでの時間調節する。
+|-
+| align="center"| '''[[Team:Chiba/Project/Experiments:Signal Molecule Quencher|Exp #1 Partial quenching of signals (jamming)]]'''
+| align="center"| '''[[Team:Chiba/Experiments:copy number|Exp #2 Balancing Player]]'''
+| align="center"| '''[[Team:Chiba/Experiments:LuxR_mutant|Exp #3 Lux Mutants]]'''
+| align="center"| '''[[Team:Chiba/Project/Experiments:Sender_Crosstalk|Exp #4 Spoken to by Foreigners]]'''
+| align="center"| '''[[Team:Chiba/Project/Experiments:Receiver_Crosstalk|Exp #5 Speaking to Foreigners]]'''
-調節する方法には、二つが挙げられる。
-*蓄積するときの割合を変える。
-*閾値自体を変える。
-*右の絵は一定の割合で水がある容器の中にたまっていく様子を示している。
-*ここでは、容器に水がたまることった時が閾値を超えた時とする
-*一番上は蓄積するスピードが速く、容器が小さいのでたまる時間は短い
-*真ん中は蓄積するスピードは速いが、容器が大きいためにたまる時間は遅くなる
-*一番下は蓄積するスピードが遅く、容器が大きいためにたまる時間がより遅くなる
-<br　clear=all>
-==System design==
-１、全体の説明
-蓄積したシグナル分子によって遺伝子発現が起こる細胞間コミュニケーションである[[Team:Chiba/about_qs|クオラムセンシング]]を利用する。
-クオラムセンシングではLuxI protein familyがシグナル分子であるAHLを合成する。
-LuxR protein familyはAHLに応答し、Lux promoter下の遺伝子を発現する。
-LuxI protein familyによって合成されたAHLを蓄積していく->ある点(応答閾値)まで蓄積すると、遺伝子発現が起こる
-合成されたAHLが応答閾値に達するまでの時間を変えることで、遺伝子発現までの時間を調整することができる。
-２、どうやって？
-応答閾値を変える方法はいくつか考えられる
-・合成するシグナルを変える
-・シグナルを受け取るレシーバーを変える
-・シグナル自体を分解し、応答しにくくする
-GFPを発現させ、蛍光強度によって遺伝子発現を調べる
-==Experiments and Result==
-===Signal Molecule Sender ===
-[[Image:Chiba project design Sender.jpg|left]]
-　English:Each species has their own LuxI-type proteins,which synthesize their specific autoinducers,AHLs.AHLs produced by different LuxI-type proteins differ only in the length of the acyl-chain moiety and substitution at position C-3.LuxR,which is original for Vibrio fischeri,is activated by its cognate autoinducer,3OC6HSL.However,LuxR is also activated by non-endogenous molecules,C4HSL,C6HSL,and 3OC12HSL.Activation by non-endogenous molecules requires a higher signal concentration[http://partsregistry.org/Part:BBa_F2620:Specificity <sup>(2)</sup>].This results in slower activation of receivers,when AHL concentration is increasing.日本語:異なる生物はそれぞれに異なるLuxIタイプのタンパク質を持ち、アシル鎖の長さ、あるいはC-3位の置換基が異なる種類のAHLを合成する。それぞれの生物種のLuxIタイプのタンパク質、それが合成する分子名は以下の表のようである。
-(Fig.4).AHLを受け取り応答するLuxRタンパク質は''Vibrio fischeri''由来であり、3OC6HSLに応答する。しかし、他種生物由来のAHLにも応答することが知られており、このとき、より高い濃度のAHLが必要となる[http://partsregistry.org/Part:BBa_F2620:Specificity <sup>(2)</sup>].AHLがゆっくり溜まっていく時、LuxRは3OC6HSLに対して最も早く応答し、他のAHLに対してはそれよりも遅く応答する。
-(冨永)
-====[[Team:Chiba/Sender experiments#Design|more about sender design]]====
-<br clear=all>
-====Crosstalk test====
-[[image:senders_crosstalk_chiba_01.gif|frame|left|
-'''Fig.''' senders crosstalk test.senders strain XL10Gold,Receiver strain JW1908.Reaction temparature was 30°C.All measurements are averages from three replicate cultures with error bars representing standard deviations.Labeling:LuxI,RhlI,LasI means fluorescence induced by AHLs synthesized by LuxI,RhlI,LasI respectively.]]
-*軸ラベル、直します。
-*RhlIとLuxIでは、GFP inductionにかかる時間はほとんど同じであった.
-*LasIは、GFP inductionが他より約2時間遅れた.
-（冨永）
-====[[Team:Chiba/Sender experiments#Experiment|more about Sender experiment and result]]====
-<br clear=all>
-===Signal Molecule Receiver ===
-[[Image:Chiba project design Receiver.jpg|left]]
-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。
-#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.
-#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.
-#レシーバーのコピーナンバーを変える.
-（福冨、小林）
-<br clear=all>
-{| style="border:0px;" cellpadding="5px"
-| width="30%" valign="top" |
-====[[Team:Chiba/Receiver#crosstalk|Quorum-Sensing Crosstalk]]====
-レシーバーたんぱくを変えて、クロストークを起こさせる<br>
-[[Image:Receiver switch chiba.jpg|frame|left|'''Fig.　''' クロストーク]]<br>
-| width="30%" valign="top" |
-====[[Team:Chiba/Receiver#copy_number|Plasmid Copynumber]]====
-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を変えることで、応答までの時間を変える<br>
-[[Image:Receiver copy number chiba.jpg|frame|left|'''Fig.''' copynumber]]<br>
-| width="30%" valign="top" |
-====[[Team:Chiba/Receiver#LuxR_mutant|LuxR mutant]]====
-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
-[[Image:Receiver switch mLuxR chiba.jpg|frame|left|'''Fig.''' LuxR mutant]]
 |}
+</center>
-<!--====Quorum-Sensing Cross-talk====
+*'''Others'''
-[[Image:Receiver switch chiba.jpg|left]]
+**'''[[Team:Chiba/Experiments:Reporter|Searching for Reporters]]'''
-[[Image:AHL original pairing chiba.jpg|frame|right|'''Fig.''' original pairing ]]
+**'''[[Team:Chiba/Demo_experiments|Demonstrations]]'''
-English:
-日本語::異なる生物はそれぞれに異なるLuxRタイプのタンパク質を持ち、それぞれアシル鎖の長さ、あるいはC-3位の置換基が異なる種類のAHLに応答する。生物種によって異なるAHLと、それに応答するLuxRタイプのタンパク質は以下の表のよう。
-Pseudomonas aeruginosa由来のRhlR,LasRも3OC6HSLを受け取ることがわかっており、このとき、より高い濃度の3OC6HSLが必要となる<sup>()</sup>.3OC6HSLがゆっくり溜まっていく時、LuxRが最も早く応答し、RhlR、LasRはそれよりも遅く応答する。
-<table width="500" border="1" cellpadding="0" cellspacing="0" bordercolor="#000000" center>
-<tr>
-<td width="200">Strain</td>
-<td width="200">AHL</td>
-<td width="150">LuxR-type protein<td>
-</tr>
-<tr>
-<td>P.aeruginosa</td>
-<td>C4HSL</td>
-<td>RhlI</td>
-</tr>
-<tr>
-<td>V. fisheri</td>
-<td>3OC6HSL</td>
-<td>LuxI</td>
-</tr>
-<tr>
-<td>P.aeruginosa</td>
-<td>3OC12HSL</td>
-<td>LasI</td>
-</tr>
-</table>
-(冨永)
+===Our (incomplete) metamorphosizing image ===
+<br>
+[[Image:Demo-flower Chiba.gif|frame|left|'''Fig.4a''' Flower should have blossomed!!!<br>
+Leaves, a stem and a flower was drawned with [http://partsregistry.org/Part:BBa_T9002 BBa_T9002], T9002-p15A, and AiiA Receiver, respectively on the plate containing [http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL sender)]--->more about [[Team:Chiba/Demo_experiments#Demo Experiment ~Temporal imaging system~|Temporal imaging system Demo experiments detail]]]]
 <br clear=all>
-====Copy number of Receiver Plasmid====
+==Conclusion==
-[[Image:Receiver copy number chiba.jpg|left]]
+In conclusion, we tried (and are trying) to device a series of delay-switches by designing the "switching consortia". We got limited, by certain success in generating delay switches.
-<br>English:
+#We confirmed that various "foreign" AHL sender can activate the LuxR/ LuxP switch.
-<br>日本語:レシーバーのコピーナンバーを減らすことで、GFPが確認できるまでの時間を延長する。
+#Using the 3OC12HSL (Las-type) instead of 3OC6HSL (Lux-type) as signaling input, we could delay the activation of the LuxR/ LuxP switch for 2 hours. As of today, Oct 29th), this is the slowest delay switch in our hand.
-コピーナンバーが少なくなれば、LuxRの合成量は減少する。
+#Interestingly, we kept failing to observe the crosstalk between Lux-sender and receivers from other organisms. Interestingly, we never seen the function of cinI/ cinR from Rhizobium leguminosarum. We have no idea why we could not reconstruct the communication (by this natural pairs).
-LuxRの合成量が減少すれば、AHLを受け取るLuxRは従来より減ってしまう。
+#Several other strategies have been tested, too. We have positive expectation especially on the engineering of LuxR protein.
-そのため、プロモーターの活性化は遅くなり、GFPの発現量は減る。
+#Fiinally, we have provided 13 biobricks that will be useful in various future projects using cell-cell communications.
-したがって、コピーナンバーが多いレシーバーより、GFPが確認できるまでの時間を延長させることができる。
-（杉山）
-<br clear=all>
+One of the technical challenge of our project was that the reporter genes are quite slow by itself to develop readable signal. There are significant time-lag between transcription initiation and the time the reporter start emitting the signal. We have screened many different reporters including lucifeases, lacZ, and other fluorescent proteins. With our experimental setup, they all looked more or less the same level (some were in the range of 30-60 mins in microscope, but all needed 90-120 mins to get visible by eye). For more precise PoPS analysis, we should have conduct blotting analysis, instead of fluorescent analysis.
-====LuxR/Plux mutants show====
+Biological timer was previously designed by [https://2007.igem.org/Missouri_Miners Missouri Miners (2007)]. In their system, they feed arabinose (input signal) to the bacteria, which consume the signal molecule. Upon eating up the molecule, the bacteria will generate GFP, signaling that time is up. This system is clever in that one can freely set the timer by adjusting the amount of arabinose fed to the cells. Our approach is more to make a series of timers. Obvious drawback of this approach is that we have to make new timer for each and every applications. Good thing about our system is that once created, we can run several timers simultaneously in a single pot; Combinatorial use of biological timers with different delay time would allows us to sequential switching of various genes, enabling more sophisticated control of the biological processes.
-[[Image:Receiver switch mLuxR chiba.jpg|left]]
-<br>English:
-<br>日本語:私たちはmutated Receiverを用いることで、AHL感受性の違う２種類（WTと変異体）のレシーバーを用意し、delay-timeのバリエーションを増やした。
-#a greater response to 3OC6HSL[http://authors.library.caltech.edu/5553/ <sup>(3)</sup>]
+==References==
-#a increase in sensitivity to 3OC12HSL[http://mic.sgmjournals.org/cgi/content/abstract/151/11/3589  <sup>(4)</sup>].
-<br clear=all>
-====[[Team:Chiba/Receiver_experiments|Receiver experiments details]]====
--->
-===Signal Molecule Quencher===
-[[Image:Chiba project design.jpg|left]]
-AiiAによるAHL分解を同時に起こすことで、AHL供給速度を遅くする.
-*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.
-====[[Team:Chiba/AiiA_experiments|AiiA experiments detail]]====
-<br clear=all>
-==Demo Experiments==
-実験内容とdataかるく
-====[[Team:Chiba/Demo_experiments|Demo experiments detail]]====
-English:
-<BR>日本語:
-<BR>
-シグナルを受け取るレシーバーを変える(クロストークの利用),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、そしてレシーバータンパク質であるLuxRに変異を入れることによりreceiverのGFPの発現までにずれが生じるはず。
-それを目視で確認しよう！
-<BR>
-・確認の仕方
-<BR>
-℃で培養しているreceiverに時間(30min?)ごとにUVをあててGFPが見えるかチェックする。
-<BR>
-香取
-==Conclusion==
-けつろん
-==Future Work==
-==references==
 #[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]
 #[http://partsregistry.org/Part:BBa_F2620:Specificity BBa_F2620:Specificity]
+#[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]
+#[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]
 #[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]
 #[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]
-{| style="color:white;background-color:Maroon" cellpadding="3" cellspacing="3" border="1" bordercolor="white" width="100%" align="center"
+#[http://mic.sgmjournals.org/cgi/content/full/153/12/3923 Paul Williams :Quorum sensing, communication and cross-kingdom signalling in the bacterial world.Microbiology 153 (2007), 3923-3938]
+#[http://pubs.acs.org/cgi-bin/abstract.cgi/acbcct/2006/1/i11/abs/cb6004245.html D. J. Sayut ''et al.:''Construction and Engineering of Positive Feedback Loops.ACS Chemical Biology.'''1'''.No.11.(2006)]<br>
+#[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WBK-4PRHJ6K-2&_user=136872&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=136872&md5=3ed7ffab9f831d102f5e99353843c080 D. J. Sayut ''et al.:''Noise and kinetics of LuxR positive feedback loops.Biochem. Biophys. Res. Commun.'''363'''(3),2007,667-673.]
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