Team:Chiba/Project

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:<Font Size="7">'''''E''.coli Time Manager '''</Font>  <html><a href="http://www.chiba-u.ac.jp/e/" target="_blank"><img src="https://static.igem.org/mediawiki/2009/5/5f/Logo_chiba-u.gif" align="right" width="250"></a></html>
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!align="center"|[[Team:Chiba|Home]]
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!align="center"|[[Team:Chiba/Team|The Team]]
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!align="center"|[[Team:Chiba/Reference|Reference]]
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!align="center"|[[Team:Chiba/Notebook|Notebook]]
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!align="center"|[[Team:Chiba/Notebook/protocol|Protocols]]
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!align="center"|[[Team:Chiba/Acknowledgements|Acknowledgements]]
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!align="center"|[[Team:Chiba/Contact|Contact]]
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'''[[Team:Chiba/Project|The Project]]'''
'''[[Team:Chiba/Project|The Project]]'''
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*[[Team:Chiba/Project#Introduction|Introduction]]
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'''1,''' [[Team:Chiba/Project#Introduction|Introduction]]
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*[[Team:Chiba/Project#Project_Design|Project Design]]
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'''2,''' [[Team:Chiba/Project#Project_Design|Project Design]]
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*[[Team:Chiba/Project#Conclusions|Conclusions]]
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'''3,''' [[Team:Chiba/Project#Experiments,_Results_&_Discussion|Experiments, Results & Discussion]]
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'''[[Team:Chiba/Project/Delay_Switch|Delay Switch]]'''
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*[[Team:Chiba/Project/Delay_Switch#Experiments_(2008)|Experiments 2008]]
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3-1, [[Team:Chiba/Project#Making_LuxR_Mutants|Making LuxR Mutants]]
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*[[Team:Chiba/Project/Delay_Switch#Results_(2008)|Results 2008]]
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*[[Team:Chiba/Project/Delay_Switch#Experiments_(2009)|Experiments 2009]]
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*[[Team:Chiba/Project/Delay_Switch#Results_(2009)|Results 2009]]
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'''[[Team:Chiba/Project/Parallel|Parallel Activation]]'''
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*[[Team:Chiba/Modeling|Modeling]]
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*[[Team:Chiba/Project/Parallel#Experiments|Experiments]]
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*[[Team:Chiba/Project/Parallel#Results|Results]]
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'''[[Team:Chiba/Project/Series|Series Activation]]'''
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*[[Team:Chiba/Modeling|Modeling]]
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3-2, [[Team:Chiba/Project#Characterization_of_LuxR_Mutants|Characterization of LuxR Mutants]]
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*[[Team:Chiba/Project/Series#Experiments|Experiments]]
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3-3, [[Team:Chiba/Project#Demonstration|Demonstration]]
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*[[Team:Chiba/Project/Series#Results|Results]]
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'''5,''' [[Team:Chiba/Project#Conclusions|Conclusions]]
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<Font Size="6">''E''.coli Time Manager Since 2008</Font>  <html><a href="http://www.chiba-u.ac.jp/e/" target="_blank"><img src="https://static.igem.org/mediawiki/2009/5/5f/Logo_chiba-u.gif" align="right" width="250"></a></html>
 
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!align="center"|[[Team:Chiba|Home]]
 
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!align="center"|[[Team:Chiba/Team|The Team]]
 
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!align="center"|[[Team:Chiba/Parts|Parts]]
 
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!align="center"|[[Team:Chiba/Reference|Reference]]
 
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!align="center"|[[Team:Chiba/Notebook|Notebook]]
 
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!align="center"|[[Team:Chiba/Notebook/protocol|Protocols]]
 
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!align="center"|[[Team:Chiba/Links|Links]]
 
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!align="center"|[[Team:Chiba/Acknowledgements|Acknowledgements]]
 
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!align="center"|[[Team:Chiba/Contact|Contact]]
 
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__NOTOC__
__NOTOC__
== '''Introduction''' ==
== '''Introduction''' ==
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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.
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=== Implementing a "Timer" Function! ===
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== Project Design ==
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For making some timers without need of synchronizer system and that can be used in a single system we try to make following design.
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===複数細胞の同時コントロール===
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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.
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==== Signaling System ====
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In this project, we use acylated homoserine lactones (AHLs), signaling
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molecules used for [http://en.wikipedia.org/wiki/Quorum_sensing quorum sensing] in gram negative bacteria.
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'''Senders''' express LuxI or similar enzymes, which catalyze the production
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of AHLs, under the control of a constitutive (Tet) promoter. Each cell
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thus generates AHL more or less at a constant rate. AHL can freely
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permeate cell membranes and are detected by neighboring cells.
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'''Receivers''' constitutively express LuxR proteins (or a similar
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ortholog), the protein that detects AHL concentrations. When AHLs bind
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LuxR proteins, the AHL-LuxR complex activates the Lux promoter. The
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threshold [AHL] at which switching occurs is determined by the
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affinity of AHL for the particulr LuxR ortholog.
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[https://2008.igem.org/Team:Chiba/about_qs|(more about quorum sensing)]
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=== Constructing A Delay Switch (Since 2008) ===
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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.
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==== Constructing A Delay Switch, Multiple Ways ====
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In principle, there are three ways to delay the activation of chemical communications;
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#'''Silencing the Speakers''': Rate of signal accumulation down-regulated, for instance, by slowing down the signal generators.
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#'''Desensitize Receivers''': Switching threshold elevated, for instance, by using insensitive receiver/ reporter systems.
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#'''Partial Blocking''': Decreasing the by chewing the signal up.
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'''Inter-species communications!'''
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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/Delay_Switch#Exp_.234_Spoken_to_by_Foreigners|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/Delay_Switch#Exp_.235_Speaking_to_Foreigners|See exp #5]].
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:::::::'''[[Team:Chiba/Project/Delay_Switch|-> Get more information about a Delay Switch!]]'''
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=== Constructing A Flow of Activation ===
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*Our project is to make a sort of "timer", where gene activation is triggered after a certain time.
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*Our approach to this goal is to create a series of transcription factors (TFs) that can be activated by the same inducer molecule but with a different sensitivity. In an environment where the inducer concentration gradually levels up, these TFs switches on one by one according to the order of sensitivity.
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*We believe such collection of such TF variants would be useful for the timing control in biological function at will, and thereby contribute to the synthetic biology & iGEM community.
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*Using a set of  TF variants, we aimed to draw an "animated picture": a picture that pop up one by one.
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<embed  width="320" height="220"  src="https://static.igem.org/mediawiki/2009/b/bd/Chiba_parallel_and_series.SWF?autostart=true&amp;repeat&amp;https://static.igem.org/mediawiki/2009/b/bd/Chiba_parallel_and_series.SWF" quality="high" type="application/x-shockwave-flash"  allowfullscreen="true" />
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<embed  width="300" height="220"  src="https://static.igem.org/mediawiki/2009/1/17/Chiba_demonstration.SWF?autostart=true&amp;repeat&amp;https://static.igem.org/mediawiki/2009/1/17/Chiba_demonstration.SWF" quality="high" type="application/x-shockwave-flash"  allowfullscreen="true" />
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Fig. 1 Completion drawing of our bacterial timer
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We are trying to make a platform for generating an animated pictures using series of new timer cells we have constructed.
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'07のMissouri Minersや'08の私たちのプロジェクトは、シンクロシステムの必要がないタイマーを作ることに成功しましたが、1種類の系では1種類のタイマーしか使えないという欠点がありました。私たちは同一系内で複数のタイマーの起動をさせようと考えています。
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この欠点を補うために考えたのがMutantを利用したParallel Activation Systemと、Crosstalkがおこらない組み合わせを利用したSeries Activation Systemです。
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== Project Design ==
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[[Image:Chiba LuxR.png|frame|left|Fig. 2 Function of LuxR :(1)LuxR protein generation, (2)AHL binding domain, (3)dimerization, (4)DNA binding domain]]
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*To create such a timer, we utilized LuxR, a protein used to mediate [[Team:Chiba/Project/Signaling-system|cell-cell communication during quorum sensing]], as shown in the figure on the left.
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*By inserting point-mutations in the ''luxR'' gene, we sought to create LuxR proteins requiring a variety of lengths of time to activate downstream transcription.
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<br style="clear: both" />
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== Experiments, Results & Discussion ==
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===[[Making delayed-LuxR mutants|Making delayed-LuxR mutants]]===
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====Experiments====
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[[Image:Chiba_making_Luxr.jpg|frame|center|Fig.3 Y Directed evolution to get some delayed-LuxR mutants]]
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*We created a mutant library of LuxR (size = 8000) with the use of error-prone PCR and incorporated the resulting coding regions into expression vectors.
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*The vectors were then transformed into a strain of E.coli JW1226 cells harboring plux-gfp and grew approximately 200 colonies.
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*The colonies were lifted off the agar plate with a nitrocellulose filter and transfered to a plate containing AHL in order to observe the evolution of GFP fluorescence over time (Fig. 4).
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*We selected 13 colonies that were slow to display fluorescence, which we designated as delayed-LuxR mutants.
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[[Image:chiba09_exp2.png|frame|center|Fig. 4 display fluorescence]]
<center>
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==== [[Team:Chiba/Project/Parallel|Parallel Activation]] ====
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<embed  width="300" height="220"  src="https://static.igem.org/mediawiki/2009/0/09/Chiba-by_pentax.SWF?autostart=true&amp;repeat&amp;https://static.igem.org/mediawiki/2009/0/09/Chiba-by_pentax.SWF" quality="high" type="application/x-shockwave-flash"  allowfullscreen="true" />
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Parallel Activationとは数種類のLuxR Mutantを使ったシステムである。
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別々のLuxR Mutantが入った細胞を、同時に培養し、蛍光蛋白を順々に発現させようというものである。
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異なる種類の細胞タイマーを同時に起動させるということから、私たちはこのシステムをParallel Activationと呼んでいる。
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対訳:
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=== Characterization of LuxR Mutants ===
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The Parallel Activation is a system that uses several varietys of LuxR Mutants. The Cells containing different Mutants that is differ each other by virtue of time of responce are cultured in one same system and express the fluorescent proteins in turn. We call this system "Parallel Activation" because of using various Mutants concurrently.
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[[Image:Chiba-LuxRs characterization.png|frame|right|Fig.5 Characterization of LuxR Mutants]]
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==== Experiments (Fig.5) ====
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#JW1226  bacterial strains co-transformed with plasmids containing either wild-type or mutant (13 variants) LuxR and pLux-GFP were cultured at  37&deg;C for 12h.
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#Using a pin, the culture was transfered to a nitrocellulose filter, placed on an agar plate, cultured at 37&deg;C for an additional 12h.
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#The nitrocellulose filter was then transfered to a solid medium containing 0, 1, 10, 100, or 1000 nM AHL.
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#Designating this the start point, we observed fluorescence through the naked eye at 30 minute intervals for 4 hours under UV light of 365nm (Funakoshi UVGL-58 at long wave mode).
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<br style="clear: both" />
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==== Fluorescence scored at near-end point (6h) ====
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[[Image:Chiba-Mutants3.png|frame|center|Fig.6 Fluorescence photos at 6h after incubation]]
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*Mutant LuxRs showed different threshold in AHL concentration required for switching.
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*As expected, many (not all) of the mutants isolated in our screening were with lower sensitivity to AHL, compared to WT: Mutant #2 and #4 showed similar threshold value of [AHL] with WT. Most others requied about 10x higher concentration for switching.  Mutants #1, #12, and #13 required as much as 100x higher concentration of AHL (1uM).
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*The result indicates that one of the major 'solution' to generate delay in color development  was to down-tune the sensitivity of LuxRs.
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<br style="clear: both" />
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====  Time-course color development on the plate with fixed (100 nM) conc. of AHL. ====
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[[Image:Chiba-Mutants4.png|center|frame|Fig.7 fluorescence photos on 100 nM AHL medium plate]]
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<br style="clear: both" />
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*Upon full induction, the intensity of GFP fluorescence reaches plateau in 90 mins: this gives the time-resolution of our animated picture.
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*At the end point of this experiment (6h after induction), clone #1 and #2 showed same level of fluorescence (blitheness) with WT. However, they were significantly slower in color development. Note that the switching threshold of clone #2 appears almost the same with WT at the end point (fig.6). This indicates that this clone is as sensitive to AHL as wildtype but less efficient in activating the LuxP promoter, thereby realizing the apparent delay in our system.
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*Many others exhibited slower color development, and they did not reach the maximum level of fluorescence. Further analysis (transfer curves in different time points) would provide more insight into the molecular basis on how they achieve the delayed color development.
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'''[[Team:Chiba/Project/Parallel|-> Get More Information about "Parallel Activation"]]'''
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====Genotypes====
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*The following lists where point-mutations were incorporated into the ''luxR'' gene.
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<center>
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[[Image:Chiba-Mutants.png|frame|center|Fig. 8 Hypothetical positions of residues in TraR corresponding to those found to modulate acyl-HSL specificity in LuxR(Nassser et al., 2007). The crystal structure of the LuxR homologue TraR (PDB 1L3L) has been determined (Zhang et al., 2002). Pink represents AHL-binding domain and cyan represents DNA-binding domain.]]
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*A delayed GFP expression (in comparison to wild-type LuxR) phenotype was observed from colonies housing genes in which either the AHL-binding domain or the DNA binding domain had been mutated.
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==== [[Team:Chiba/Project/Series|Series Activation]] ====
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=== Demonstration ===
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</center>
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#Double-Transformed (pUC-LuxR Mutants series-ColE1 and pLux-GFPuv-P15A) and cultured on LB-Amp-Cm-plate. (37°C,12h)
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We use several types of cell-cell signaling system for Series Activation. At the first switch, a signal molecule is secreted. When the signaling by the molecule occurs, cells that received the molecule express a fluorescence protein and next different signaling molecule. Then next cells receive second molecule and express another fluorescence protein and third signal molecule. Continuing such processes, we meke time-delay syssem for timer. Parallel Activation system is, so to speak, a "whisper down the lane" system.
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#Pick and cultured in 10mL of LB-Amp-Cm (37°C,12h).
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#Drawing picture with those liquid culture on nitrocellulose Filter.
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#Those nitrocellulose Filter placed on LB-Amp-Cm-plate.
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#Cultured at 37°C, 12h.
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#Transfer pictures on [[Team:Chiba/Project/for_painting|special]] plate.
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#Exposed to UV (365nm) light once every 30 minutes to observe GFP fluorescence.
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<gallery>
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Image:Chiba_DSCF6003.jpg|T=0
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Image:Chiba_DSCF6019.jpg|T=130 min
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Image:Chiba_DSCF6023.jpg|T=150 min
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Image:Chiba_DSCF6029.jpg|T=180 min
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Image:Chiba_DSCF6039.jpg|T=230 min
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Image:Chiba_DSCF6046.jpg|T=260 min
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Image:Chiba_DSCF6054.jpg|T=300 min
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Image:Chiba_DSCF6060.jpg|T=360 min
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</gallery>
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'''[[Team:Chiba/Project/Series|-> Get More Information about "Series Activation"]]'''
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*For the determination of best condition for E coli painting, [[Team:Chiba/Project/for_painting|Click here]]!
== Conclusions ==
== Conclusions ==
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*By using error-prone PCR, we have created a LuxR library.
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*With a simple and convenient screening method, we have isolated various LuxR mutants which confer delayed switching behavior in GFP signals.
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*By introducing these LuxR variants together with reporter genes (such as GFP) under the control of Lux promoter, we created bacteria 'ink's that develop their color with unique delay-time.
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*We conducted painting with these bacteria inks thereby created animated pictures.
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*Characterization of LuxR mutants is ongoing. But our preliminary data showed that some of the variants turned out to be the one with less sensitivity to AHLs, and others seemed to be as sensitive as wild-type LuxR but seemed less efficient somewhere in the downstream process.
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*We created variety of Biobricks during the course of this projects. Some of them are characterized and sent to the HQ, and many more are almost ready for shipping!
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<br /><br /><br />
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*[http://chem.tf.chiba-u.jp/igem/2009demo.html see more pics?]
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Latest revision as of 04:00, 22 October 2009

E.coli Time Manager


The Project

1, Introduction

2, Project Design

3, Experiments, Results & Discussion

3-1, Making LuxR Mutants

3-2, Characterization of LuxR Mutants

3-3, Demonstration

5, Conclusions

Introduction

Implementing a "Timer" Function!

  • Our project is to make a sort of "timer", where gene activation is triggered after a certain time.
  • Our approach to this goal is to create a series of transcription factors (TFs) that can be activated by the same inducer molecule but with a different sensitivity. In an environment where the inducer concentration gradually levels up, these TFs switches on one by one according to the order of sensitivity.
  • We believe such collection of such TF variants would be useful for the timing control in biological function at will, and thereby contribute to the synthetic biology & iGEM community.
  • Using a set of TF variants, we aimed to draw an "animated picture": a picture that pop up one by one.

Fig. 1 Completion drawing of our bacterial timer

Project Design

Fig. 2 Function of LuxR :(1)LuxR protein generation, (2)AHL binding domain, (3)dimerization, (4)DNA binding domain
  • To create such a timer, we utilized LuxR, a protein used to mediate cell-cell communication during quorum sensing, as shown in the figure on the left.
  • By inserting point-mutations in the luxR gene, we sought to create LuxR proteins requiring a variety of lengths of time to activate downstream transcription.


Experiments, Results & Discussion

Making delayed-LuxR mutants

Experiments

Fig.3 Y Directed evolution to get some delayed-LuxR mutants
  • We created a mutant library of LuxR (size = 8000) with the use of error-prone PCR and incorporated the resulting coding regions into expression vectors.
  • The vectors were then transformed into a strain of E.coli JW1226 cells harboring plux-gfp and grew approximately 200 colonies.
  • The colonies were lifted off the agar plate with a nitrocellulose filter and transfered to a plate containing AHL in order to observe the evolution of GFP fluorescence over time (Fig. 4).
  • We selected 13 colonies that were slow to display fluorescence, which we designated as delayed-LuxR mutants.
Fig. 4 display fluorescence


Characterization of LuxR Mutants

Fig.5 Characterization of LuxR Mutants

Experiments (Fig.5)

  1. JW1226 bacterial strains co-transformed with plasmids containing either wild-type or mutant (13 variants) LuxR and pLux-GFP were cultured at 37°C for 12h.
  2. Using a pin, the culture was transfered to a nitrocellulose filter, placed on an agar plate, cultured at 37°C for an additional 12h.
  3. The nitrocellulose filter was then transfered to a solid medium containing 0, 1, 10, 100, or 1000 nM AHL.
  4. Designating this the start point, we observed fluorescence through the naked eye at 30 minute intervals for 4 hours under UV light of 365nm (Funakoshi UVGL-58 at long wave mode).


Fluorescence scored at near-end point (6h)

Fig.6 Fluorescence photos at 6h after incubation
  • Mutant LuxRs showed different threshold in AHL concentration required for switching.
  • As expected, many (not all) of the mutants isolated in our screening were with lower sensitivity to AHL, compared to WT: Mutant #2 and #4 showed similar threshold value of [AHL] with WT. Most others requied about 10x higher concentration for switching. Mutants #1, #12, and #13 required as much as 100x higher concentration of AHL (1uM).
  • The result indicates that one of the major 'solution' to generate delay in color development was to down-tune the sensitivity of LuxRs.


Time-course color development on the plate with fixed (100 nM) conc. of AHL.

Fig.7 fluorescence photos on 100 nM AHL medium plate


  • Upon full induction, the intensity of GFP fluorescence reaches plateau in 90 mins: this gives the time-resolution of our animated picture.
  • At the end point of this experiment (6h after induction), clone #1 and #2 showed same level of fluorescence (blitheness) with WT. However, they were significantly slower in color development. Note that the switching threshold of clone #2 appears almost the same with WT at the end point (fig.6). This indicates that this clone is as sensitive to AHL as wildtype but less efficient in activating the LuxP promoter, thereby realizing the apparent delay in our system.
  • Many others exhibited slower color development, and they did not reach the maximum level of fluorescence. Further analysis (transfer curves in different time points) would provide more insight into the molecular basis on how they achieve the delayed color development.


Genotypes

  • The following lists where point-mutations were incorporated into the luxR gene.
Fig. 8 Hypothetical positions of residues in TraR corresponding to those found to modulate acyl-HSL specificity in LuxR(Nassser et al., 2007). The crystal structure of the LuxR homologue TraR (PDB 1L3L) has been determined (Zhang et al., 2002). Pink represents AHL-binding domain and cyan represents DNA-binding domain.
  • A delayed GFP expression (in comparison to wild-type LuxR) phenotype was observed from colonies housing genes in which either the AHL-binding domain or the DNA binding domain had been mutated.

Demonstration

  1. Double-Transformed (pUC-LuxR Mutants series-ColE1 and pLux-GFPuv-P15A) and cultured on LB-Amp-Cm-plate. (37°C,12h)
  2. Pick and cultured in 10mL of LB-Amp-Cm (37°C,12h).
  3. Drawing picture with those liquid culture on nitrocellulose Filter.
  4. Those nitrocellulose Filter placed on LB-Amp-Cm-plate.
  5. Cultured at 37°C, 12h.
  6. Transfer pictures on special plate.
  7. Exposed to UV (365nm) light once every 30 minutes to observe GFP fluorescence.


  • For the determination of best condition for E coli painting, Click here!

Conclusions

  • By using error-prone PCR, we have created a LuxR library.
  • With a simple and convenient screening method, we have isolated various LuxR mutants which confer delayed switching behavior in GFP signals.
  • By introducing these LuxR variants together with reporter genes (such as GFP) under the control of Lux promoter, we created bacteria 'ink's that develop their color with unique delay-time.
  • We conducted painting with these bacteria inks thereby created animated pictures.
  • Characterization of LuxR mutants is ongoing. But our preliminary data showed that some of the variants turned out to be the one with less sensitivity to AHLs, and others seemed to be as sensitive as wild-type LuxR but seemed less efficient somewhere in the downstream process.
  • We created variety of Biobricks during the course of this projects. Some of them are characterized and sent to the HQ, and many more are almost ready for shipping!




  • [http://chem.tf.chiba-u.jp/igem/2009demo.html see more pics?]