Team:Chiba/Project

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<Font Size="7">'''''E''.coli Time Manager '''</Font> <Font Size="5">-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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:<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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'''[[Team:Chiba/Project|The Project]]'''
'''[[Team:Chiba/Project|The Project]]'''
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#[[Team:Chiba/Project#Introduction|Introduction]]
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#[[Team:Chiba/Project#Project_Design|Project Design]]
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'''1,''' [[Team:Chiba/Project#Introduction|Introduction]]
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#[[Team:Chiba/Project#Experiments,_Results_&_Discussion|Experiments, Results & Discussion]]
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##[[Team:Chiba/Project#Making_LuxR_Mutants|Making LuxR Mutants]]
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'''2,''' [[Team:Chiba/Project#Project_Design|Project Design]]
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##[[Team:Chiba/Project#Characterization_of_LuxR_Mutants|Characterization]]
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</td></tr><tr><td>
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##[[Team:Chiba/Project#For_Improving_Pictures|For improving pictures]]
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'''3,''' [[Team:Chiba/Project#Experiments,_Results_&_Discussion|Experiments, Results & Discussion]]
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##[[Team:Chiba/Project#Demonstration|Demonstration]]
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</td></tr><tr><td>
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#[[Team:Chiba/Project#Conclusions|Conclusion]]
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3-1, [[Team:Chiba/Project#Making_LuxR_Mutants|Making LuxR Mutants]]
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__NOTOC__
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3-2, [[Team:Chiba/Project#Characterization_of_LuxR_Mutants|Characterization of LuxR Mutants]]
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</td></tr><tr><td>
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3-3, [[Team:Chiba/Project#Demonstration|Demonstration]]
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</td></tr><tr><td>
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'''5,''' [[Team:Chiba/Project#Conclusions|Conclusions]]
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__NOTOC__
== '''Introduction''' ==
== '''Introduction''' ==
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=== Implementing a "Timer" Function! ===
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|style="width:80%;"|* our project is to make a "bacterial timer" i.e. ~~(もっとぐたいてきに).
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* our approach to this goal is to make a series of a transcription factor which each of them differes in a responce time(?) of the transcription activation(?) by an single(same?) inducer.
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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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* we believe that this device would be useful for making an macroscopic(?) timing control(?) in bacterial behavior or many application in synthetic biology & iGEM community.
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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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* to demonstrate this "timing control", we aimed(?) to draw an "animated picture": a picture that pop up (emerge?) one by one.  
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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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*上の様な感じはどうですか?英語に自信はない --[[User:Maiko|Maiko]] 12:47, 20 October 2009 (UTC)
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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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Fig. 1 Completion drawing of our bacterial timer
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(手術予定)
 
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*細胞通信を使ったBacterial Timer を創ります。
 
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*E. coli Timer完成予想図(画像作成中です--[[User:Yoshimi|Yoshimi]] 09:12, 20 October 2009 (UTC))
 
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*Timerを創る方法は様々(diffusion of molecules, switching(oscillator, communication, arabinose, plac, etc... ))であるが、なぜ細胞通信を選んだのか。
 
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そして、昨年は2段階の通信しか創れなかった。バリエーションが乏しい。
 
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*作戦変更!(Sender側をかえたCrosstalk通信にはバリエーションを創るのに限界があるので、今年はReceiver側の調節をする。)目指すは時計の数字分の、12通りの時間差をつくりたい!
 
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*
 
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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.
 
== Project Design ==
== 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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(手術予定)
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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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2) Project Design
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⑤Receiver側のAHL通信の仕組みを図説する。(1,AHLが入るところ(培地調節) 2,Receiver中のLuxR 3,Receiver中のReporter)
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==== Signaling System ====
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*なんじゃこりゃーなおします。--[[User:Yoshimi|Yoshimi]] 13:22, 20 October 2009 (UTC)
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*sender使わないからいらないのでは?というか,cell-cell comunicationは使ってなくてただのtranscription activatorとしてluxRを使っているだけ...という面がつよい...ので,luxRの転写活性化がどのようにおこるか,をメインにしてはいかが?--[[User:Maiko|Maiko]] 13:43, 20 October 2009 (UTC)
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*次項のスクリーニングでsenderを使うので、ここでも図にSenderを入れておくべきだと考えます。--[[User:Yoshimi|Yoshimi]] 14:10, 20 October 2009 (UTC)
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[[Image:Chiba_quorumsensing.gif|600px]]
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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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== Experiments, Results & Discussion ==
== Experiments, Results & Discussion ==
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===[[Making delayed-LuxR mutants|LuxR mutantづくり]]===
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===[[Making delayed-LuxR mutants|Making delayed-LuxR mutants]]===
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*directed evolutionで色々な応答速度のLuxR mutantをつくることを目指した。
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====Experiments====
====Experiments====
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[[Image:chiba09_exp.png|frame|center|Fig. Y Directed evolution to get some delayed-LuxR mutants]]
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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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*error-prone PCRでLuxRの変異ライブラリを作製し,発現ベクターに組み込んだ
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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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*これを,E.coli BW(?) harboring plux-gfpに形質転換し,コロニーを形成させた。
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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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*コロニーをニトロセルロース膜でリフトし,AHL入りのプレートにのせ,gfpの蛍光の経時変化をみた(Fig. X)
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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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*蛍光しはじめるのが遅いものを13個pickし,delayed-LuxR変異体を得た。
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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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*pickした13個のクローンの転写活性化速度?を,再度transformして確認し,最終的に○個の速度バリエーションのluxR変異体を得た。
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[[Image:chiba09_exp2.png|frame|center|Fig. 4 display fluorescence]]
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[[Image:chiba09_exp2.png|frame|center|Fig. X LuxRライブラリ?によるのPlux-gfp蛍光経時変化]]
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*(こんな感じでよいでしょか?絵はとりあえずポスタのやつを画面キャプチャしてベタばり --[[User:Maiko|Maiko]] 13:09, 20 October 2009 (UTC))
 
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(手術予定)
 
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⑥Mutantを創る。(Error-proneが良い理由:バリエーションを創りやすい。新たなBiobrick作成方法としてError-prone PCRとスクリーニングをセットにしMutant-Partsの作り方を説明する。)
 
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⑦さらに詳しい実験方法(Biobrickからerror-prone PCRをおこなったことなど)
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=== Characterization of LuxR Mutants ===
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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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⑧得られたデーター(なぜ8000のライブラリを、200に絞り、どうやって13sampleを選び出したのか説明)
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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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*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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=== 得られたLuxR mutantのcharacterization ===
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⑨LuxR Mutant 個性確定実験の実験方法
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⑩100 nM培地での応答の遅れについての結果
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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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⑪AHL濃度を振った場合の、6h後の蛍光強度の差についての結果
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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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⑫LuxR Mutantの個性と、変位が入っている部分からの考察
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=== より良い絵を描くために/To draw more better picture ===
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*we screened the "agar media condition" (the thickness & the agar conc.) to make difference in the delay of the LuxR mutant more bigger(appearent?).
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*ちょっと思ったのだがこのAHL濃度とか培地とかふりまくってるのを載せると,LuxRの時間差が見えた理由が「LuxR変異体のおかげ」というよりは「培地のおかげ」っぽくなるので,この培地スクリーニングは実は載せない(というかちっちゃくのせとくのは良いけどVIP待遇はしない)方がまだ恰好がつくのでは...^w^;--[[User:Maiko|Maiko]] 14:05, 20 October 2009 (UTC)
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*”より差をみせるため”の実験なのでここでは軽く説明し、あとはリンクつけます。
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*⑬培地を振った実験について。実験方法と結果。
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'''培地のよりよい条件を見つける'''
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私たちはデモをよりはっきり見せるために必要な培地の条件を調べた。
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まず、次の4種類の培地を作成した。
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1)agar 1% and 薄い(約1.5 mm)
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2)agar 5% and 厚い(約7 mm)
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3)agar 1% and 薄い(約1.5 mm)
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4)agar 5% and 厚い(約7 mm)
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この4種類の培地上にNitro Cellulose filterをのせ、その上でそれぞれ13種のMutantsを3 coloniesずつ培養した。
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培養後、Nitro Cellulose filterごとそれぞれの培地をカッターで切り出して同じ厚さの2000 nMのAHLを含む培地にのせて目視で蛍光の観察を始めた。
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この実験をした結果、各mutant LuxRが光りだす時刻の差は、(4)のagar 5% and 厚い 培地で最も大きい差が見られた。
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つまり厚さが薄かったりagar が少なかったりした培地上でのmutant LuxRが光りだす時刻のばらつき具合よりも、この(4)の培地上でのばらつき具合の方がはっきりしていた。
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だから私たちはこの培地条件を使ってdemoをすることにした。
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*⑭Reporter毎の応答の差についての実験と結果。GFPuvがDelayを見やすい。
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私たちは、ReporterとしてGFPuv, sfGFP, mRFP, mCherry, mOrangeを使って上に挙げたものと同じ実験をした。
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*⑮Reporter毎の応答についての考察。
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=== Demonstration ===
=== Demonstration ===
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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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#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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⑯E. coli Timer完成デモ
 
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(⑰できればアニメ)
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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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手術予定
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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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18・Biobrickを使って(iGEM的には)新たな方法(error-prone PCR)を用い、新パーツをつくりました。
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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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・何通り(ただいま確認中)のバリエーションの遅れを生み出せました。(Mutant+WT:4~5種、gel調節:2種、Reporters: 4通り・・・これらの組み合わせの分(タイミングがかぶるのは除く)だけ時間差をうみだせた!)
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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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・demonstrationをおこないました。
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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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⑲ Future Works : Whisper down the lane
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時計というものに、時間だけでなく順番も入れられる。
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例えば培地中に拡散するAHLにムラがあったとしても、ちゃんと順番通りに動くシステム、
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バリエーションは作れないが順番ならば得意。
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成長するアニメーションを作るには、この順番通りに動くシステムが必要。
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アニメを作るときは時間の刻み方が人にとって意味を持つようなものであることが大切。
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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?]