Chiba/Quorum Sensing

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[[Image:chiba_logo.png|center]]
[[Image:chiba_logo.png|center]]
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{| style="border:0;width:100%;font-family:'Trebuchet MS'" cellpadding="20px" cellspacing="0"
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[[Chiba|Introduction]] | [[Chiba/Project_Design|Project Design]] ( [[Chiba/Engeneering_Flagella|1.Sticky Hands]] | [[Chiba/Communication|2.Communication]] | [[Chiba/Quorum_Sensing|3.Size Control]] ) |  [[Chiba/Making Marimo|Making Marimos]] |  [[Chiba/Goal|Our Goal]] || [[Chiba/Team_Members|Team Members]] | [[Chiba/Members_Only|メンバ連絡簿]]
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[[Chiba|Home]]<br>
 +
<span style="font-size:120%;font-weight:bold;">[[Chiba/Introduction|Introduction]] | [[Chiba/Project_Design|Project Design]] ( [[Chiba/Engeneering_Flagella|1.Affinity Tag]] | [[Chiba/Communication|2.Communication Module]] | [[Chiba/Quorum_Sensing|3.Size Control]] ) |  [[Chiba/Making Marimo|Making Marimos]] |  [[Chiba/Goal|Our Goal]]</span><br>
 +
[[Chiba/Acknowledgements|Acknowledgements]] | [[Chiba/Team_Members|Team Members]] | [http://chem.tf.chiba-u.jp/igem/ iGEM Chiba Website] | [[Chiba/Members_Only|メンバ連絡簿]]  
|}
|}
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==Size Control: Our Aim==
+
==Size Control==
-
[[Image:Chiba_proj_ahlconc.png|frame|'''Fig1.'''AHL concentration gradient.]]
+
===Our Aim===
-
(マリモのサイズをコントロールしたい/しなくてはならない:どっち?)マリモのサイズをコントロールする為にはAHLの届く範囲をコントロールできればよい.
+
[[Image:Chiba_proj_ahlconc.png|frame|'''Fig. 15''' Controlling AHL diffusing area and the size of Bacteria Marimo.]]
-
*AHLの届く範囲をコントロールするために以下の3つの方法を考えた:
+
Since produced AHL diffuses all around the bacteria culture, all receivers can ultimately aggregate to one huge marimo with their sticky hands.  This final state is not the case of real marimos.  Thus our idea for controlling the size of Bacteria Marimos is based on the high performance quorum sensing or AHL-diffusing inhibition.
-
*#SenderのAHL生産能力をあげる
+
#Raise the AHL productivity of Sender
-
*#ReceiverのAHL感受性をあげる
+
#Increase the AHL sensitivity of Receiver
-
*#AHL分解酵素aiiAを使う
+
#Use the AHL degrading enzyme aiiA to localize AHL
<br clear="all">
<br clear="all">
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===Design===
===Design===
-
AHL is synthesizing from Met via metK/luxR.<br>
+
AHL is synthesized from methionine by the enzyme MetK and LuxI.<br>
-
We thought combined overexpression of these 2 genes enhances the sender capacity.
+
We tested in the hope of combined overexpression of these 2 genes will enhance the sender capacity.
-
AHLはFig.2のpathwayで合成されている.AHLを増加させるためにMetKを過剰発現させ,methionineを加えてみる。
+
<br>[[Image:Chiba_ahlsynth.png|frame|'''Fig. 16''' Passway synthesizing AHL in bacteria.]]
-
<br>[[Image:Chiba_ahlsynth.png|frame|Fig.2]]
+
<br clear="all">
<br clear="all">
===Experiment===
===Experiment===
-
[[Image:Chiba AHL test 02.jpg]]
+
[[Image:Chiba AHL test 02.jpg|frame|'''Fig. 17''' Testing procedure of sender capability.]]
 +
====Sender====
 +
Ptet-LuxI
 +
*Synthesize AHL constantly
 +
 
 +
====metK Sender====
 +
[[Image:metK circuit.jpg]]
 +
*Synthesize AHL / express metK (precursor supplier) constantly
 +
 
 +
====Receiver====
 +
[[Image:BBa T9002.jpg|350x48px]]
 +
*BBa T9002 (Express GFP in response to AHL)
 +
 
 +
====Method(Fig3.)====
 +
#Transform sender (Ptet-luxI) and MetK sender (pLac-luxI-metK) into E coli strains (XL10Gold and BW25113)
 +
#Inoculated them independently in liquid media.  Incubated at 37℃ 12h.
 +
#Checked OD<sub>600</sub>, washed, and diluted senders to adjust cell number (5x10<sup>8</sup>,5x10<sup>7</sup>,......, 5x10<sup>2</sup>).
 +
#Mix with receiver (pLux-GFP/ BW25113, 1.5x10<sup>9</sup> cells)
 +
#Shake for 1-3h at room tempature (optonal; methionine (10mM) added)
 +
#Spindowned and fluorescence checked.
===Result===
===Result===
-
[[Image:AHL test photo 01.jpg|320x240px]]
+
[[Image:AHL test photo 01.jpg|frame|left|'''Fig. 18''' Testing minimum amount of senders required for activating receivers.<br> ]]
-
[[Image:AHL test photo 02.jpg|240x320px]]
+
<br clear="all">
 +
*At least 5x10<sup>4</sup> cell/ml sender required to activate lux promoter in BW25113.
 +
*Sender performance HIGHLY depends on the strains: 100x more cells were required to activate the lux promoter in other strain XL10Gold. 
 +
*Overexpression of metK did not result in the booster effect. It was so even when Methionine (10mM), the direct substrate of metK, was added to the growth media (not shown). Sucks.
===Discussion===
===Discussion===
-
*合成されたS-adenosyl Methionineがほかの代謝経路に使われて、AHL合成に使われなかったのだろうか.
+
*No improbement in sending power was observed.
 +
*Assumption (not conclusion):<br>
 +
(1) Precursor supply (S-adenosyl methionine) is simply at enough level, and luxR is by far the rate-limiting step in AHL synthetic pathway. No chance/ need for metabolic engineering of this pathway. <br>
 +
(2) Precursor flows out to somewhere else. Or overexpression of metK ativates the degradation pathway of its product. Anyway too hard for us to cope with in short time.
 +
<br>
 +
*Note; We haven't confirmed the entire sequencing of the metK.
-
 
+
==2.Sensitizing Receiver==
-
==2.Improving Receiver==
+
===Design===
===Design===
-
[[Image:mut LuxRの感度アップグラフ]]
+
[[Image:MLuxRx.gif|frame|left|'''Fig. 19''' Transfer curve of LuxR mutant to AHL.''' Data modified from Collins ''et.al.'' Mol. Microbiol. 55, 712–723 (2005)''']][[Image:MLuxRxxx.gif|frame|left|'''Fig. 20''' LuxR mutations focused in our project ]]<br clear="all">
-
mutated LuxRによって感度が上がったという論文があった[ref].論文によれば1点にmutantをいれた場合wildtypeにくらべ10nMでのGPF発現量が増加する.3点にmutantをいれた場合wildtypeのGFP発現の限界が10nMに対して、1.5nMでGFPを発現することができるようになる。
+
Collins ''et.al.'' described the hyper-sensitive variants of luxR to AHL.(Collins, C. H., Arnold, F. H. & Leadbetter, J. R. Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones. ''Mol. Microbiol.'' '''55''', 712–723 (2005))<br>
-
私たちは3つのMutationをそれぞれ一つずつ入れたときは、3つのうち2つがGFPの発現量が多くなっているという理由から,そのうちの1点mutantと2点mutantを作った.
+
We created two luxR mutants. One is I14F mutant of LuxR, and another is I45F/S116A.<br>
 +
There is no report on I45F/S116A double mutant.By creating/ analyzing this mutant in AHL sensitivity, we can tell the effect of T33A in LuxR.<br>
===Experiment===
===Experiment===
-
[[Image:作ったmut luxr]]
+
[[Image:MLuxRxx.gif|frame|'''Fig. 21''' Location of the mutations in luxR gene]]<br>
-
図のように1点&2点にミュータントをいれたluxrをsite direct mutagenesisで作成した.
+
luxR mutants was analyzed in their sensitivity to AHL by following procedure.<br>
-
これをBBa_t9002プラスミドにいれた.
+
 
-
AHLの入ったプレートにより感度を見た.また、蛍光リーダを用いて、10nMでの発現測定した。
+
#Inoculate Receiver (wild type luxR/BW25113), mutated Receiver (1point mutation/BW25113) in liquid media for 12 h at 37℃.
 +
#Adjust the cell density and add AHL (final concentration, 0, 0.1, 1, 5, 10, 100, and 1000 nM)
 +
#Incubate for 1h at room temperature
 +
#Spindown cells and GFP folurescent level checked<br><br><br>
===Result===
===Result===
-
*Ile45->Phe,Ser116->Alaの2か所の変異を入れたものは常にGFPを発現し続けるために、Swichingができなくなってしまった。AHLを入れずにGFPを発現。
+
[[Image:MLuxR-test.gif|frame|left|'''Fig. 22''' comparison 1point mutated and wild type]]
-
[[Image:Two mutation.jpg|320x240px]]
+
*Both wild type and 1point mutation luxR express GFP under 5nM AHL concentration.
 +
<br><br><br><br><br><br><br><br><br>
 +
[[Image:Two mutation.jpg|320x240px|frame|left|'''Fig. 23''' LuxR mutant with two point mutations expressed GFP without adding AHL]]
 +
*2point mutation(I45F,S116A) luxR Receiver express GFP without AHL.
 +
<br><br><br><br><br><br><br><br><br><br><br><br><br><br>
-
*一か所変異を入れたものはwild typeのLuxRを発現するものよりもGFPの発現が多くなった。
+
===Discussion===
 +
[[Image:MluxR_table.jpg |frame|left|'''Table 1  Seisitivity of LuxR mutants.''' Data modified from Collins ''et.al.'' Mol. Microbiol. 55, 712–723 (2005)]]<br><br><br><br><br><br><br><br><br>
 +
 
 +
 
 +
This table is quoted from paper.<br>
 +
In the paper, DH5α strain was used.<br>
 +
But wild type luxR in our study expressed GFP under 5nM AHL concentration.<br>
 +
 
 +
Assumption:There is some difference between strains.
 +
Required to do same test on other strains
 +
 
 +
==3.Localizing AHL==
-
==3.Aiia==
 
-
(なんかいいタイトルないかな)
 
===Design1===
===Design1===
-
[[Image:Plux aiiA.jpg|402x45px]]<br>
+
[[Image:BBa I729006 circuit.jpg]]<Br>
-
図のように,AHLのシグナルが入るとaiiaを合成するような回路を考えた.Marimo全体がAHL quencherとして働く.Marimoの内側でAHLを分解し、Marimoの外側のAHL濃度を抑える.
+
BBa I729006<br>
 +
In addition to the receiver unit, there added AiiA under the control of lac promoter.<br>
 +
AiiA degrades/ inactivates AHL, lowering the AHL concentration in the cell. <br>
 +
This way cells installed with this circuit can respond, but less sensitive, to the sender cells.
 +
 
====Experiment====
====Experiment====
 +
#Inoculate E.coli carries this aiiA receiver plasmid in Liquid Media.
 +
#At the same time, inoculate AHL sender E.coli in Liquid Media.
 +
#Dilute receiver cells and spread on agar plate, and spot sender 1μL. Incubate at 37℃ 12hour
 +
#Check plate.(GFP expressed?)
====Result====
====Result====
-
*BBa_T9002との比較ではGFPははじめ発現しないが、しばらくするとGFPが発現するようになる。
+
GFP expression was not observed on the plate of aiiA receiver.
 +
 
 +
[[Image:T9002 plate.jpg|frame|'''Fig. 24''' Receiver|left]]
 +
[[Image:AiiA cons plate.jpg|frame|'''Fig. 25''' aiiA receiver|center]]
 +
<br clear="all">
 +
 
====Discussion====
====Discussion====
-
*発現し始めた時が、AHLの濃度がaiiAの分解る能力を超えた時だと考えられる。
+
We thought expression of aiiA significantly decrease the cellular level of AHL. It turned out to be true.<br>
 +
However, it went too much. It does not respond to senders anymore, probably due to too fast AHL degradation. <br>
 +
We need more controlled/ moderate expression of aiiA.
===Design2===
===Design2===
 +
[[Image:Plux aiiA.jpg|402x45px]]<br>
 +
This gene circuit is expected so that aiiA is synthesized only when the receiver senses AHL signal.  If so, the whole Bacteria Marimo becomes AHL quencher.  In fact, the bacteria inside of the Marimo degrades AHL and the AHL diffusion outside of Marimo is inhibited.
 +
====Experiment====
 +
#Inoculate E.coli carries this aiiA receiver plasmid in Liquid Media.
 +
#At the same time, inoculate AHL sender E.coli in Liquid Media.
 +
#Dilute receiver cells and spread on agar plate, and spot sender 1μL. Incubate at 37℃ 12hour
 +
#Put plate 30℃
 +
#Check GFP expression every 1hour.
 +
 +
====Result====
 +
*When we compared the GFP expression with the test tube of BBa_T9002, the bacteria with the consructed gene circuit did not fluoresce immediately after incubation, however fluoresced in 18~20 hour.<br>
 +
 +
====Discussion====
 +
*GFP expression means plux is activated. So we could make moderate aiiA receiver.
 +
*But we need more twisted circuit.
 +
 +
===Design3===
[[Image:Rec inv aiia.jpg|402x45px]]<br>
[[Image:Rec inv aiia.jpg|402x45px]]<br>
-
図のようにインバーターをかませた.高い濃度ではaiiAが発現しないためAHLは分解されない.AHLが低い濃度ではaiiAが発現しAHLを分解する.よってマリモの中心付近ではAHLを分解せず,外側へ行くと分解される.
+
Above picture describes aiiA is regulated inverted lux promoter with CI inverter.
 +
*High AHL concentration: no aiiA expression, no AHL degrade.
 +
*Low AHL concentration: aiiA expressed, AHL degraded.
 +
 
 +
 
 +
 
====Experiment====
====Experiment====
-
このパーツを作ろうとしたが、inverterを組み合わせた時点で終わってしまった。
+
We could not finish assembling this part. It will be our future work.
 +
 
 +
Subpart:BBa_S03840
 +
 
 +
[[Image:S03840.jpg]]

Latest revision as of 05:41, 27 October 2007

Chiba logo.png

Home
Introduction | Project Design ( 1.Affinity Tag | 2.Communication Module | 3.Size Control ) | Making Marimos | Our Goal
Acknowledgements | Team Members | [http://chem.tf.chiba-u.jp/igem/ iGEM Chiba Website] | メンバ連絡簿


Size Control

Our Aim

Fig. 15 Controlling AHL diffusing area and the size of Bacteria Marimo.

Since produced AHL diffuses all around the bacteria culture, all receivers can ultimately aggregate to one huge marimo with their sticky hands. This final state is not the case of real marimos. Thus our idea for controlling the size of Bacteria Marimos is based on the high performance quorum sensing or AHL-diffusing inhibition.

  1. Raise the AHL productivity of Sender
  2. Increase the AHL sensitivity of Receiver
  3. Use the AHL degrading enzyme aiiA to localize AHL


1.Improving Sender

Design

AHL is synthesized from methionine by the enzyme MetK and LuxI.
We tested in the hope of combined overexpression of these 2 genes will enhance the sender capacity.


Fig. 16 Passway synthesizing AHL in bacteria.


Experiment

Fig. 17 Testing procedure of sender capability.

Sender

Ptet-LuxI

  • Synthesize AHL constantly

metK Sender

MetK circuit.jpg

  • Synthesize AHL / express metK (precursor supplier) constantly

Receiver

BBa T9002.jpg

  • BBa T9002 (Express GFP in response to AHL)

Method(Fig3.)

  1. Transform sender (Ptet-luxI) and MetK sender (pLac-luxI-metK) into E coli strains (XL10Gold and BW25113)
  2. Inoculated them independently in liquid media. Incubated at 37℃ 12h.
  3. Checked OD600, washed, and diluted senders to adjust cell number (5x108,5x107,......, 5x102).
  4. Mix with receiver (pLux-GFP/ BW25113, 1.5x109 cells)
  5. Shake for 1-3h at room tempature (optonal; methionine (10mM) added)
  6. Spindowned and fluorescence checked.

Result

Fig. 18 Testing minimum amount of senders required for activating receivers.


  • At least 5x104 cell/ml sender required to activate lux promoter in BW25113.
  • Sender performance HIGHLY depends on the strains: 100x more cells were required to activate the lux promoter in other strain XL10Gold.
  • Overexpression of metK did not result in the booster effect. It was so even when Methionine (10mM), the direct substrate of metK, was added to the growth media (not shown). Sucks.

Discussion

  • No improbement in sending power was observed.
  • Assumption (not conclusion):

(1) Precursor supply (S-adenosyl methionine) is simply at enough level, and luxR is by far the rate-limiting step in AHL synthetic pathway. No chance/ need for metabolic engineering of this pathway.
(2) Precursor flows out to somewhere else. Or overexpression of metK ativates the degradation pathway of its product. Anyway too hard for us to cope with in short time.

  • Note; We haven't confirmed the entire sequencing of the metK.

2.Sensitizing Receiver

Design

Fig. 19 Transfer curve of LuxR mutant to AHL. Data modified from Collins et.al. Mol. Microbiol. 55, 712–723 (2005)
Fig. 20 LuxR mutations focused in our project

Collins et.al. described the hyper-sensitive variants of luxR to AHL.(Collins, C. H., Arnold, F. H. & Leadbetter, J. R. Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones. Mol. Microbiol. 55, 712–723 (2005))
We created two luxR mutants. One is I14F mutant of LuxR, and another is I45F/S116A.
There is no report on I45F/S116A double mutant.By creating/ analyzing this mutant in AHL sensitivity, we can tell the effect of T33A in LuxR.

Experiment

Fig. 21 Location of the mutations in luxR gene

luxR mutants was analyzed in their sensitivity to AHL by following procedure.

  1. Inoculate Receiver (wild type luxR/BW25113), mutated Receiver (1point mutation/BW25113) in liquid media for 12 h at 37℃.
  2. Adjust the cell density and add AHL (final concentration, 0, 0.1, 1, 5, 10, 100, and 1000 nM)
  3. Incubate for 1h at room temperature
  4. Spindown cells and GFP folurescent level checked


Result

Fig. 22 comparison 1point mutated and wild type
  • Both wild type and 1point mutation luxR express GFP under 5nM AHL concentration.










Fig. 23 LuxR mutant with two point mutations expressed GFP without adding AHL
  • 2point mutation(I45F,S116A) luxR Receiver express GFP without AHL.















Discussion

Table 1 Seisitivity of LuxR mutants. Data modified from Collins et.al. Mol. Microbiol. 55, 712–723 (2005)










This table is quoted from paper.
In the paper, DH5α strain was used.
But wild type luxR in our study expressed GFP under 5nM AHL concentration.

Assumption:There is some difference between strains.
Required to do same test on other strains

3.Localizing AHL

Design1

BBa I729006 circuit.jpg
BBa I729006
In addition to the receiver unit, there added AiiA under the control of lac promoter.
AiiA degrades/ inactivates AHL, lowering the AHL concentration in the cell.
This way cells installed with this circuit can respond, but less sensitive, to the sender cells.

Experiment

  1. Inoculate E.coli carries this aiiA receiver plasmid in Liquid Media.
  2. At the same time, inoculate AHL sender E.coli in Liquid Media.
  3. Dilute receiver cells and spread on agar plate, and spot sender 1μL. Incubate at 37℃ 12hour
  4. Check plate.(GFP expressed?)

Result

GFP expression was not observed on the plate of aiiA receiver.

Fig. 24 Receiver
Fig. 25 aiiA receiver


Discussion

We thought expression of aiiA significantly decrease the cellular level of AHL. It turned out to be true.
However, it went too much. It does not respond to senders anymore, probably due to too fast AHL degradation.
We need more controlled/ moderate expression of aiiA.

Design2

Plux aiiA.jpg
This gene circuit is expected so that aiiA is synthesized only when the receiver senses AHL signal. If so, the whole Bacteria Marimo becomes AHL quencher. In fact, the bacteria inside of the Marimo degrades AHL and the AHL diffusion outside of Marimo is inhibited.

Experiment

  1. Inoculate E.coli carries this aiiA receiver plasmid in Liquid Media.
  2. At the same time, inoculate AHL sender E.coli in Liquid Media.
  3. Dilute receiver cells and spread on agar plate, and spot sender 1μL. Incubate at 37℃ 12hour
  4. Put plate 30℃
  5. Check GFP expression every 1hour.

Result

  • When we compared the GFP expression with the test tube of BBa_T9002, the bacteria with the consructed gene circuit did not fluoresce immediately after incubation, however fluoresced in 18~20 hour.

Discussion

  • GFP expression means plux is activated. So we could make moderate aiiA receiver.
  • But we need more twisted circuit.

Design3

Rec inv aiia.jpg
Above picture describes aiiA is regulated inverted lux promoter with CI inverter.

  • High AHL concentration: no aiiA expression, no AHL degrade.
  • Low AHL concentration: aiiA expressed, AHL degraded.


Experiment

We could not finish assembling this part. It will be our future work.

Subpart:BBa_S03840

S03840.jpg