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 ([http://en.wikipedia.org/wiki/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 [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.^(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 [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 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.

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]
[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)]
[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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 __NOTOC__
 ==Abstract==
-====''E.coli'' time manager====
+====　''E. coli'' time manager====
-We are constructing the delay switches to control/ preset the timing of 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 various ‘switching consortium’ with a variety of preset time.
+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 [[Team:Chiba/Project#references|(1),(2)]]. Communication using non-endogenous molecules is less sensitive than the original, 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:Project design chiba QS.gif|frame|right|'''Fig.1''' Project design]]
+[[Image:Chiba_popcorn.jpg|thumb|right|'''Fig. 1　Burnt popcorn'''. He could have prevent this if he properly preset the timer...]]
-VCR systems possess the time-recording function. Microwaves automatically stop heating when the right time comes. Using their '''presetting''' functions, we became free either from staying up late watching European succor game, and from worrying about our popcorn burned black while we are yelling to the game videotaped. This way, the timer functions have revolutionized our lifestyle.
+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, hopefully 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 can program much more [[Team:Chiba/Project/Applications|complex behaviors]] in cellular systems.
+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.
-As one of the thousands of possible applications, we are trying to construct '''temporal imaging system''' using ''E. coli'' 'ink's that differ not in color but the 'timing' of coloration (fig).  Over time, parts of images (or characters) are getting visible one by one, making animated message/ picture. In the end, the last ink get colorized, covering the entire image. Such system should be useful as a sort of secured communication board:  we can convey our message to those who know the exact moment they should take a look. After a while, all the massage is gone.
+[[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 Details==
+==Project Design==
-私たちは，２種類のbacteriaからなる Switching consortium によって，水時計型のtime delay装置をつくる(more about [http://en.wikipedia.org/wiki/Water_clock Water clock-wikipedia.en])  。
+[[Image:Chiba fig3 2.png|right|frame|'''Fig. 3　System design''']]
-まず，Sender細胞が，ゆっくりとシグナルを合成する。それは次第に系に蓄積する。Receiverの応答感度に達した時，Receiverは遺伝子機能を起動するわけである（図説）。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、遺伝子発現時間を調整することができる。そしてそのtime delay（preset time）は，sender側のmessage蓄積速度と，receiverの応答感度を調節すれば，任意に設定できる。
-===Signaling System===
+We designed a '''switching consortium''' that works like a water clock
-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。そもそもクオラムセンシングは，自分たちの密度がある閾値が一定値を超えたときに起動するものである（→もっと詳しく）。
+([http://en.wikipedia.org/wiki/Water_clock Water clock-wikipedia.en]). Here is how it works (Fig 3).
-Sender：LuxI protein familyがシグナル分子であるAHLを合成する。この分子は細胞膜を自由に通過し，Neighbering cellsにも感知される。
-Receiver：常時LuxR proteinを発現し，AHLを監視している。AHL濃度がある値を超えると結合し，Lux promoter下の遺伝子発現をONにする。その応答閾値はLuxRとAHLとの親和性に依存している（文献）。
+. 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.
-===Delayed switches, four ways:===
+. 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.
-==Project Details==
+. Either by changing the receiver sensitivity or rate of signal
-[[Image:System design Chiba 1.jpg|right|frame|'''Fig. ''' System design]]
+accumulation, one can freely control the delay length of the
-１、全体の説明
+individual switches. Using switches with various times-of-delay, one
+can sequentially activate many different cellular functions.
-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。
+===Signaling System===
-LuxR protein familyはAHLに応答し、Lux promoter下の遺伝子を発現する。
+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)]]
-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
+===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.
-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。
+'''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.]]
-応答閾値までAHLが蓄積すると、GFPを発現する
+==List of Experiments==
+===='''For details, click each of the index titles'''====
-その蛍光強度によって遺伝子発現を調べる
+<center>
+{| style="border:0px;" cellpadding="0px" |
+|-
+| 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''' ]]
+|-
-<br clear=all>
+| 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]]'''
-２、How to control the timing of gene expression
+| 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]]'''
-[[Image:Whole_system_design_Chiba.jpg|center]]
-{| style="border:0px;" cellpadding="5px"
-| width="30%" valign="top" align="center"|
-[[Image:Chiba project design Sender.jpg]]<br clear=all>
-'''[[Team:Chiba/Project#Signal Molecule Sender Phase|Signal Molecule Sender Phase]]'''
-| width="30%" valign="top" align="center"|
-[[Image:Chiba project design Receiver.jpg]]<br clear=all>
-'''[[Team:Chiba/Project#Signal Molecule Receiver Phase|Signal Molecule Receiver Phase]]'''
-| width="30%" valign="top" align="center"|
-[[Image:Chiba project design.jpg|Chiba project design.jpg]]<br clear=all>
-'''[[Team:Chiba/Project#Signal Molecule Quencher|Signal Molecule Quencher]]'''
 |}
+</center>
+*'''Others'''
+**'''[[Team:Chiba/Experiments:Reporter|Searching for Reporters]]'''
+**'''[[Team:Chiba/Demo_experiments|Demonstrations]]'''
+===Our (incomplete) metamorphosizing image ===
-===Quorum Sensing Cross-talk  ===
-クオラムセンシングにおける送受信装置は由来する生物ごとに特有のセットをなしているが、異種の送受信装置同士もCross-talkすることが分かっている。
-Cross-talkによるコミュニケーションは感度が鈍いため、Receiverの活性化が遅くなる。
-====[[Team:Chiba/Qurum Sensing Cross-talk|more about Quorum Sensing Cross-talk ]]====
-==Experiments and Result==
-===Signal Molecule Sender Phase===
-====Design====
-[[Image:Chiba project design Sender.jpg|left]]
-'''Utilize Quorum Sensing Cross-talk'''
-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[[Team:Chiba/Project#references|<sup>(1),(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[[Team:Chiba/Project#references|<sup>(1),(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 AHL sender phase design]]
-<br clear=all>
-====Result====
-'''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.]]
-*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 Phase ===
-[[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.
-<br clear=all>
-{| style="border:0px;" cellpadding="5px"
-| width="50%" valign="top" |
-====[[Team:Chiba/AHL Receiver Phase#crosstalk|Quorum-Sensing Crosstalk]]====
-[[Image:Receiver switch chiba.jpg|frame|left|'''Fig.　''' Crosstalk]]<br clear=all>
-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。<br>
-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[[Team:Chiba/Project#references|<sup>(1)</sup>]][[Team:Chiba/Project#references|<sup>(2)</sup>]]。<br>
-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。
 <br>
-[[Team:Chiba/AHL Receiver Phase#Quorum Sensing Crosstalk|more about Receiver phase crosstalk]]
+[[Image:Demo-flower Chiba.gif|frame|left|'''Fig.4a''' Flower should have blossomed!!!<br>
-[[Image:Copy-nomber-change-result Chiba.gif|frame|left|'''Fig.　''' Time Delay Test]]<br clear=all>
+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]]]]
-[[Team:Chiba/AHL Receiver Phase#Quorum Sensing Crosstalk|more about experimental result]]
-| width="50%" valign="top" |
-====[[Team:Chiba/AHL Receiver Phase#Plasmid_Copy_number|Plasmid Copynumber]]====
-[[Image:Receiver copy number chiba.jpg|frame|left|'''Fig.''' copynumber]]<br clear=all>
-遺伝子回路を含むプラスミドをもった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<br>
-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<br>
-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<br>
-[[Team:Chiba/AHL Receiver Phase#Plasmid Copy number|more about Plasmid Copy number]]<br>
-[[Image:Copynomber-change-result Chiba.gif|frame|left|'''Fig.''' Time Delay Test]]<br clear=all>
-*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
-[[Team:Chiba/AHL Receiver Phase#Plasmid_Copy_number|more about experimental result]]
-| width="50%" valign="top" |
-|}
-<!--====Quorum-Sensing Cross-talk====
-[[Image:Receiver switch chiba.jpg|left]]
-[[Image:AHL original pairing chiba.jpg|frame|right|'''Fig.''' original pairing ]]
-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)
 <br clear=all>
-====Copy number of Receiver Plasmid====
-[[Image:Receiver copy number chiba.jpg|left]]
-<br>English:
-<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。
-コピーナンバーが少なくなれば、LuxRの合成量は減少する。
-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。
-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。
-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>
-====LuxR/Plux mutants show====
-[[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>]
-#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]]====
--->
-====[[Team:Chiba/AHL Receiver Phase#LuxR_mutant|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[[Team:Chiba/Project#references|<sup>(3),(4)</sup>]]
-[[Image:Receiver switch mLuxR chiba.jpg|frame|left|'''Fig.''' LuxR mutant]]
-<br clear=all>
-===Signal Molecule Quencher===
-'''Design'''
-{| style="border:0px;" cellpadding="5px"
-| width="50%" valign="top" |
-[[Image:Chiba project design.jpg|left]]
-<br clear=all>
-*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 Receiver Phase|more about AiiA Receiver Phase]]====
-| width="50%" valign="top" |
-[[Image:AiiA-Receiver-result Chiba.gif|frame|center|'''Fig.　''' Time Delay Test]]
-<br clear=all>
-====[[Team:Chiba/AiiA Receiver Phase|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====
-結果は以下の通り。
-[[Image:Team-Chiba-demo-mihon.gif|200px]] *緑部分：LuxI　*赤部分：LasI
-[[Image:Team-Chiba-demo-1.JPG|200px]]　[[Image:Team-Chiba-demo-2.JPG|200px]]　[[Image:Team-Chiba-demo-3.JPG|200px]]
-枚目の写真は開始点。2枚目はLuxIの部分だけGFPを肉眼（蛍光灯光）で確認。３枚目はLuxIとLasI両方を確認したものです。
-（それぞれ０時間、4時間、8時間後の映像。液量が100μLと少ないため、今までの実験（1000μL）での結果よりもGFP発現に時間がかかった。・・・コレ書かない方がいいのかなあ。）
---[[User:Yoshimi|Yoshimi]] 11:04, 29 October 2008 (UTC)
--->more about [[Team:Chiba/Demo_experiments|Demo experiments detail]]
-=== Demo ~Receivers~ ===
-English:
-<BR>日本語:
-<BR>
-固体培地中にセンダー（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・・・
-・シグナルを受け取るレシーバーを変える(クロストークの利用)：LuxR,LasR,RhlR
-・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
-<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
-====Results====
---->more [[Team:Chiba/Demo_experiments|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.
+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.
-これらの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融合すれば、2時間以上の時間差の効果を期待することが出来る。
+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.
-さらにミューテーションやポジティブフィードバックループを利用することで、この時差を更に広げることが出来るだろう。
+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.
---[[User:Yoshimi|Yoshimi]] 11:09, 29 October 2008 (UTC)
+==References==
-==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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