Team:Chiba/Project/Experiments:Signal Molecule Quencher

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__NOTOC__
__NOTOC__
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==AiiA Receiver==
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==Partial quenching of signals (jamming)==
===Design===
===Design===
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'''AiiA added to Lux reporter'''
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[[Image:Chiba project design.jpg|frame|left|'''Fig. 1　Attenuating the cellular communication''' ]]
 +
We introduced AiiA [http://www.genome.jp/dbget-bin/www_bget?enzyme+3.1.1.81 (enzyme+3.1.1.81)] (the autoinducer inactivation enzyme A from ''Bacillus'') to LuxR receiver. AiiA is an enzyme that degrades AHL [[Team:Chiba/Project/Experiments:Signal Molecule Quencher#References|(1)]]  (Fig. 2). The idea was that the effective concentration of AHL in receiver cell get significantly decreased due to the constantly expressing AiiA, thereby enlarging the delay time.  Fluorescence should be observed only when [AHL] exceeds the degrading capacity of aiiA.
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[[Image:Aiia reaction chiba.gif|frame|center|'''Fig. 2　AiiA Reaction''' ]]
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'''Design'''
 
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{| style="border:0px;" cellpadding="5px"
 
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| width="50%" valign="top" |
 
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[[Image:Chiba project design.jpg|left]]
 
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| width="50%" valign="top" |
 
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'''AHL reporter with aiiA'''
 
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:Express LuxR and aiiA constantly. AiiA degrades
 
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:AHL as signaling molecule. Express GFP when
 
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:the AHL concentration exceed the capacity of aiiA.
 
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:This enables the delay of the activation time of receiver.
 
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[[Image:Aiia reaction chiba.gif|frame|left|'''Fig.''' AiiA Reaction]]
 
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<br clear=all>
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| width="50%" valign="top" |
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===Experiments===
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|}
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'''Co-transformation'''<br>
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We constructed the circuit below. AiiA is placed on the high-copy plasmid under the control of Lac promoter. It was co-transformed with the LuxR/ Plux reporter on the p15A plasmid.
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<br clear=all>
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'''Communication''' <br>
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The resultant "AiiA/LuxR reporter" was co-cultured with LuxI sender and the fluorescence was monitored over time.  As a control, we conducted the same experiment with LuxR reporter without AiiA plasmid.
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'''Detailed Condition'''
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===Experiment===
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Sender; LuxI plasmid transformed into ''E. coli'' strains (JW1908).<br>
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To make an time-delay using AiiA (an enzyme that degrades AHL), we constructed the circuit below, and compared the gfp intensity in the presence/nonpresence of AiiA.
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Receiver; LuxR-gfp plasmid transformed into ''E. coli'' strains (JW1908).<br>
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Culture/ Cndn.
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ReceiverでAiiAを共発現させて遺伝子発現を確かめる実験を行った。
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1.Both Sender and Receiver (+/- AiiA) were inoculated into small (2mL) culture and was shaken separately for 12h (at 37°C)<br>
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AHL senderとしてVibrio fischeri由来のLuxIを常に発現させ、3OC6HSLを合成させる。
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2.Inoculated into flesh media, shaken until cell density hit 2.0 in OD600<br>
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AHL Receiverとしてと,Lux promoter下にGFPを含むレポータープラスミドをダブルトランスフォーメーションさせた。
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3.Washed the cell and re-suspended. Cell density checked.<br>
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AHL senderによって合成されたAHLをAHL receiverが受取り、Lux promoter下のGFPが発現する。
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4.Mixed Sender and Receiver (Sender/Receiver 1000&mu;L/1000&mu;L).<br>
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蛍光強度を蛍光リーダーを使ってGFPの発現を計測し、遺伝子発現の活性を調べた。
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5.Incubated at 30°C.<br>
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6.Time-chased the fluorescence (485nm (excitation) and 527nm (emission)) by gfp.<br>
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使った遺伝子回路は以下の通りある。
 
{| class="tbl" |
{| class="tbl" |
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*'''Receivers'''
*'''Receivers'''
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|-
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| rowspan="2" | **[http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL autoinucer)]
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| rowspan="2" | [http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL sender)]
[[Image:LuxI-sender Chiba.gif]]
[[Image:LuxI-sender Chiba.gif]]
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===Method===
===Method===
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#Transformed Sender into E.coli strains(JW1908) and Receivers into E.coli strain(JW1908).
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#Transformed Sender into ''E. coli'' strains (JW1908) and Receivers into ''E. coli'' strain (JW1908).
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#Inoculated them independently in liquid media. Incubated at 37c&deg; 12h.
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#Inoculated them independently in liquid media. Incubated at 37&deg;C 12h.
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#Inoculated again at 37c&deg; upto about OD600=2.0
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#Inoculated again at 37&deg;C upto about OD600=2.0
#Washed them.
#Washed them.
#Mixed them (Sender:Receiver=1000&mu;l:1000&mu;l).
#Mixed them (Sender:Receiver=1000&mu;l:1000&mu;l).
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#Incubated at 30c&deg;.
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#Incubated at 30&deg;C.
#Measured intensity of green fluorescence at regular time intervals.
#Measured intensity of green fluorescence at regular time intervals.
<br clear=all>
<br clear=all>
===Result & Discussion===
===Result & Discussion===
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[[Image:AiiA-Receiver-result-detail Chiba.gif|flame|left|Fig.Time Delay Test]]
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[[Image:AiiA-Receiver-result Chiba.gif|frame|left|'''Fig. 3　Time Delay Test +AiiA Receiver vs. -AiiA Receiver''' ]]
<br clear=all>
<br clear=all>
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24時間後の蛍光強度を比較すると最大強度は1/4に下がっている。AiiAを発現させると、GFPの発現自体が大幅に減ってしまったので、AHL自体を減らしてしまうと発現量の最大値が小さくなってしまうことがわかった。つまり今回の実験ではAiiAがAHLを分解しすぎてしまっている言える。
+
The co-expression of AiiA resulted in the drastic decrease in the fluorescence all through the experiment.  It hasn't reached the endpoint even 24h after mixing. AiiA looks super-active and consume the most AHL molecule out;  Obviously, the AHL activity is way too much.
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よって本実験ではAiiA generatorがハイコピープラスミドに乗っていたので、AiiA　generatorをローコピープラスミドに乗せ換えてTime Delay Testを行いたい。
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transfer curveがシグモイドではなく、強度は低いが少なくともAHL Reporterは時間に比例して発現していた。
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On the other hand, we observed gradual increase in fluorescence over time. At least, the fluorescence from the co-culture was always above the negative control (without Lux-Sender). This indicate AiiA is not eating up the all signaling molecule. If we properly down-tune the AiiA activity (either by putting this gene into low-copy plasmid or by giving the low efficiency rbs), we should be able to see the time-delay.
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よって[http://partsregistry.org/Part:BBa_R0063 BBa_R0063　(Medium strength promoter in the absence of LuxR/HSL)]でAiiAを合成すれば、AHLが低濃度のときだけAiiAを合成し、いったんAHLがある一定の濃度を超えてしまえばAiiAによるAHL分解量は下降の一途を辿る。この遺伝子回路を用いれば新たなタイムディレイ作成の方法が提案できると考える。
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===Reference===
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===References===
#[http://www.jbc.org/cgi/content/full/279/14/13645 Wang et al.:Specificity and Enzyme Kinetics of the Quorum-quenching N-Acyl Homoserine Lactone Lactonase (AHL-lactonase).J. Biol. Chem.279(14),13645-13651,2004.]
#[http://www.jbc.org/cgi/content/full/279/14/13645 Wang et al.:Specificity and Enzyme Kinetics of the Quorum-quenching N-Acyl Homoserine Lactone Lactonase (AHL-lactonase).J. Biol. Chem.279(14),13645-13651,2004.]

Latest revision as of 09:35, 30 October 2008

Chiba-U.gif
Home The Team The Project Parts Submitted to the Registry Reference Notebook Acknowledgements


Partial quenching of signals (jamming)

Design

AiiA added to Lux reporter

Fig. 1　Attenuating the cellular communication

We introduced AiiA [http://www.genome.jp/dbget-bin/www_bget?enzyme+3.1.1.81 (enzyme+3.1.1.81)] (the autoinducer inactivation enzyme A from Bacillus) to LuxR receiver. AiiA is an enzyme that degrades AHL (1) (Fig. 2). The idea was that the effective concentration of AHL in receiver cell get significantly decreased due to the constantly expressing AiiA, thereby enlarging the delay time. Fluorescence should be observed only when [AHL] exceeds the degrading capacity of aiiA.

Fig. 2　AiiA Reaction



Experiments

Co-transformation
We constructed the circuit below. AiiA is placed on the high-copy plasmid under the control of Lac promoter. It was co-transformed with the LuxR/ Plux reporter on the p15A plasmid.

Communication
The resultant "AiiA/LuxR reporter" was co-cultured with LuxI sender and the fluorescence was monitored over time. As a control, we conducted the same experiment with LuxR reporter without AiiA plasmid.


Detailed Condition

Sender; LuxI plasmid transformed into E. coli strains (JW1908).
Receiver; LuxR-gfp plasmid transformed into E. coli strains (JW1908).
Culture/ Cndn.

1.Both Sender and Receiver (+/- AiiA) were inoculated into small (2mL) culture and was shaken separately for 12h (at 37°C)
2.Inoculated into flesh media, shaken until cell density hit 2.0 in OD600
3.Washed the cell and re-suspended. Cell density checked.
4.Mixed Sender and Receiver (Sender/Receiver 1000μL/1000μL).
5.Incubated at 30°C.
6.Time-chased the fluorescence (485nm (excitation) and 527nm (emission)) by gfp.

  • Sender
  • Receivers
[http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL sender)]

LuxI-sender Chiba.gif

  • AiiA Receiver

AiiA-Receiver-genetic-circu Chiba.gif

  • non-AiiA Receiver

Non-AiiA-Receiver Chiba.gif

Method

  1. Transformed Sender into E. coli strains (JW1908) and Receivers into E. coli strain (JW1908).
  2. Inoculated them independently in liquid media. Incubated at 37°C 12h.
  3. Inoculated again at 37°C upto about OD600=2.0
  4. Washed them.
  5. Mixed them (Sender:Receiver=1000μl:1000μl).
  6. Incubated at 30°C.
  7. Measured intensity of green fluorescence at regular time intervals.


Result & Discussion

Fig. 3　Time Delay Test +AiiA Receiver vs. -AiiA Receiver


The co-expression of AiiA resulted in the drastic decrease in the fluorescence all through the experiment. It hasn't reached the endpoint even 24h after mixing. AiiA looks super-active and consume the most AHL molecule out; Obviously, the AHL activity is way too much.

On the other hand, we observed gradual increase in fluorescence over time. At least, the fluorescence from the co-culture was always above the negative control (without Lux-Sender). This indicate AiiA is not eating up the all signaling molecule. If we properly down-tune the AiiA activity (either by putting this gene into low-copy plasmid or by giving the low efficiency rbs), we should be able to see the time-delay.

References

  1. [http://www.jbc.org/cgi/content/full/279/14/13645 Wang et al.:Specificity and Enzyme Kinetics of the Quorum-quenching N-Acyl Homoserine Lactone Lactonase (AHL-lactonase).J. Biol. Chem.279(14),13645-13651,2004.]



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