Chiba/Quorum Sensing

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< Chiba(Difference between revisions)
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(Experiment)
 
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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|Engeneering Flagella]] | [[Chiba/Quorum_Sensing|Quorum Sensing]] | [[Chiba/Goal|Our Goal]] || [[Chiba/Team_Members|Team Members]] | [[Chiba/Members_Only|メンバ連絡簿]]
+
[[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]]  
|}
|}
-
==Our Aim==
 
-
1.Not only express FliC His-tag constant constantly, apply Quorum Sensing as positive feedback genetic switch.
+
==Size Control==
-
2.Express aiiA(autoinducer inactivate enzyme A) as an AHL quencher to generate AHL concentration gradient. ?
+
===Our Aim===
-
  3.To make clear the AHL positive feedback, build genetic circuit expressing aiiA negatively on AHL concentration.  
+
[[Image:Chiba_proj_ahlconc.png|frame|'''Fig. 15''' Controlling AHL diffusing area and the size of Bacteria Marimo.]]
-
4.Improve the performance of Quorum Sensing.
+
Since produced AHL diffuses all around the bacteria culture, all receivers can ultimately aggregate to one huge marimo with their sticky handsThis 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.FliC His-tagをただ発現させるだけではなく、Quorum Sensingをpositive feedbackのスイッチとして利用する。
+
#Raise the AHL productivity of Sender
-
2.aiiAをAHL quencherとして利用し、AHLの濃度勾配をつくりだすAsseyにする。
+
#Increase the AHL sensitivity of Receiver
-
3.inverterでaiiAのthresholdをはっきり出すような遺伝子回路の設計
+
#Use the AHL degrading enzyme aiiA to localize AHL
-
4.Quorum SensingのSensing能力の向上
+
<br clear="all">
-
フィードバックじゃないのでは...?
+
==1.Improving Sender==
 +
===Design===
-
==About Quorum Sensing ==
+
AHL is synthesized from methionine by the enzyme MetK and LuxI.<br>
 +
We tested in the hope of combined overexpression of these 2 genes will enhance the sender capacity.
 +
<br>[[Image:Chiba_ahlsynth.png|frame|'''Fig. 16''' Passway synthesizing AHL in bacteria.]]
 +
<br clear="all">
 +
===Experiment===
 +
[[Image:Chiba AHL test 02.jpg|frame|'''Fig. 17''' Testing procedure of sender capability.]]
 +
====Sender====
 +
Ptet-LuxI
 +
*Synthesize AHL constantly
-
[[Image:Quorum sensing system.jpg|320x240px]]
+
====metK Sender====
 +
[[Image:metK circuit.jpg]]
 +
*Synthesize AHL / express metK (precursor supplier) constantly
-
====Sender====
 
-
AHL Sender express LuxI constantly. LuxI synthesize signaling molecule acyl-homoserine lactone(AHL). AHL go through cell membrane and diffuse into media.
 
-
SenderはLuxIを発現する。SenderはAHLを合成する。細胞膜を通り抜け細胞外にAHLは拡散していく。
 
====Receiver====
====Receiver====
-
AHL Receiver express LuxR constantly. AHL come through cell membran. AHL and LuxR bind together and activate Lux promoter.
+
[[Image:BBa T9002.jpg|350x48px]]
-
ReciverはLuxRを発現しLux promoterを含む遺伝子回路を持つ。細胞膜を通り抜けてきたAHLはLuxRと結合して、Lux promoterを活性化する。
+
*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.
-
The above genetically circuit
+
===Result===
-
上の回路では
+
[[Image:AHL test photo 01.jpg|frame|left|'''Fig. 18''' Testing minimum amount of senders required for activating receivers.<br> ]]
-
  1.The sender express luxI and synthesize AHL.
+
<br clear="all">
-
2.AHL go through cell membrane and ingest into receiver.
+
*At least 5x10<sup>4</sup> cell/ml sender required to activate lux promoter in BW25113.
-
3.AHL which ingested and LuxR conjugate to activate lux promoter.
+
*Sender performance HIGHLY depends on the strains: 100x more cells were required to activate the lux promoter in other strain XL10Gold.  
-
4.GFP which restricted by lux promoter express and turn green.
+
*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.
-
1.Sender内でLuxIが発現しAHLが合成される。
+
 
-
  2.Senderの細胞膜を通り抜けたAHLはReceiverの細胞の中に取り込まれる。
+
===Discussion===
-
  3.Receiverに取り込まれたAHLはReceiver内で発現されるLuxRと結合しlux promoterを活性化する。
+
*No improbement in sending power was observed.
-
4.lux promoter下にあるGFPが発現する。
+
*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==
 +
===Design===
 +
[[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">
 +
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>
 +
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===
 +
[[Image:MLuxRxx.gif|frame|'''Fig. 21''' Location of the mutations in luxR gene]]<br>
 +
luxR mutants was analyzed in their sensitivity to AHL by following procedure.<br>
 +
 
 +
#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===
 +
[[Image:MLuxR-test.gif|frame|left|'''Fig. 22''' comparison 1point mutated and wild type]]
 +
*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>
 +
 
 +
===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==
 +
 
 +
===Design1===
 +
[[Image:BBa I729006 circuit.jpg]]<Br>
 +
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====
 +
#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====
 +
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">
-
==Parts Assembly==
+
====Discussion====
-
====AHL Positive Feedback aiiA Receiver====
+
We thought expression of aiiA significantly decrease the cellular level of AHL. It turned out to be true.<br>
-
[[Image:Plux aiiA.jpg|402x45px]]
+
However, it went too much. It does not respond to senders anymore, probably due to too fast AHL degradation. <br>
-
====AHL Negative Feedback aiiA Receiver====
+
We need more controlled/ moderate expression of aiiA.
-
[[Image:Rec inv aiia.jpg|402x45px]]
+
-
==Experiments==
+
===Design2===
-
===AHL生産能力 Assay===
+
[[Image:Plux aiiA.jpg|402x45px]]<br>
-
====Purpose====
+
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.
-
*To know the absolute number of senders required to activate pLux.
+
====Experiment====
-
*To know difference in synthesis ability among Strains.
+
#Inoculate E.coli carries this aiiA receiver plasmid in Liquid Media.
-
*AHLのprecursurであるMethionineの有無によるAHL生産量の違い
+
#At the same time, inoculate AHL sender E.coli in Liquid Media.
-
*pLuxをアクティブにするために必要なSenderの絶対数
+
#Dilute receiver cells and spread on agar plate, and spot sender 1μL. Incubate at 37℃ 12hour
-
*株によるAHL生産能力の違い
+
#Put plate 30℃
-
*AHLのprecursurであるMethionineの有無によるAHL生産量の違い
+
#Check GFP expression every 1hour.
-
[[Image:Chiba_ahlsynth.png|frame|'''Fig.'''AHL Synthesis Pathway]]
+
-
[[Image:Chiba AHL test.jpg]]
+
====Result====
====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.
-
<Br clear="all">
+
===Design3===
 +
[[Image:Rec inv aiia.jpg|402x45px]]<br>
 +
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.
-
===To Make an AHL Concentration Gradient===
 
-
*aiiA (autoinducer inactivation enzyme A):  degradate AHLs.
 
-
*aiiAを発現させ、AHLが広がりすぎないようにする
 
-
====AHL Positive Feedback aiiA Receiver====
 
-
*pluxの下にaiiAをおき、Marimo全体がAHL quencherとして働くようにする。Marimoの内側でAHLを分解し、Marimoの外側のAHL濃度が上がらないようにする。
 
-
*普通のReceiverとともに混ぜて培養すればちょうどいい割合が見つかるかもしれない。
 
-
[[Image:Plux aiiA.jpg|402x45px]]
 
-
=====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。
 
-
*発現し始めた時が、AHLの濃度がaiiAの分解能力を超えた時だと考えられる。
 
-
====AHL Negative Feedback aiiA Receiver====
 
-
*aiiAをpluxとinverterの下に置き、AHLが低い濃度ではaiiAを発現させAHLを分解させ、高い濃度ではaiiAが発現しないような回路を作る。
 
-
**thresholdがもっとはっきりするようになるだろう。
 
-
[[Image:Rec inv aiia.jpg|402x45px]]
 
-
===Improving Sender & Receiver===
+
====Experiment====
-
====Super Sender====
+
We could not finish assembling this part. It will be our future work.
-
*SenderがもっとAHLを合成するようにする。
+
-
**AHL合成経路でS-adenosyl Methionineのをさらに合成させることでAHLの量を増やす。
+
-
metKを発現するSenderの回路を作る。
+
-
=====Result=====
+
-
あまり変化がないようである。普通のSenderと変わらない。
+
-
・原因
+
-
1.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から。
+
-
2.Genom PCRでMetKを作ったので、mutationが中に入ってしまったから
+
-
====Super Receiver====
+
Subpart:BBa_S03840
-
*Receiverをもっと高感度にする。
+
-
**論文で読んだmutant LuxRを作成し、AHLの濃度が低くてもpLuxを活性化する。
+
-
(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).)
+
-
LuxRのアミノ酸 Ile45->Phe, Ser116->Ala この変異を入れ、Sensitiveなレシーバーを作る
+
-
=====Method=====
+
-
Site direct mutagenesisでBBa_T9002に変異を入れる変異を入れる。
+
-
 LuxRに含まれるNcoI,GFPに含まれるHindⅢを利用した方法をとる。
+
-
[[Image:MLuxR.jpg]]
+
-
=====Result=====
+
[[Image:S03840.jpg]]
-
・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。
+
-
・Ile45->Pheの1か所だけの変異を入れたものを再度作ることにした
+
-
  ・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10倍ほど感度が上がった。
+

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