Team:Chiba/Project/Experiments:Signal Molecule Quencher

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We introduced [http://www.genome.jp/dbget-bin/www_bget?enzyme+3.1.1.81 AiiA](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|<sup>(1)</sup>]] (fig).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] exceed the degrading capacity of aiiA.  
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We introduced [http://www.genome.jp/dbget-bin/www_bget?enzyme+3.1.1.81 AiiA](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).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] exceed the degrading capacity of aiiA.  
[[Image:Aiia reaction chiba.gif|frame|left|'''Fig.''' AiiA Reaction]]
[[Image:Aiia reaction chiba.gif|frame|left|'''Fig.''' AiiA Reaction]]
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===Method===
===Method===
#Transformed Sender into E.coli strains(JW1908) and Receivers into E.coli strain(JW1908).
#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.
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Revision as of 00:47, 30 October 2008

Chiba-U.gif

Home The Team The Project Parts Submitted to the Registry Reference Notebook Acknowledgements


AiiA Receiver

Design

Design

Chiba project design.jpg

AiiA added to Lux reporter


We introduced [http://www.genome.jp/dbget-bin/www_bget?enzyme+3.1.1.81 AiiA](the autoinducer inactivation enzyme A from Bacillus)to LuxR receiver. AiiA is an enzyme that degrades AHL(1) (fig).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] exceed the degrading capacity of aiiA.

Fig. AiiA Reaction

--Masahiro 00:40, 30 October 2008 (UTC)

Experiments

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. --Maiko 23:31, 29 October 2008 (UTC)

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。 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。 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。 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。 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。

使った遺伝子回路は以下の通りある。

  • 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.Time Delay Test


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。 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。

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。 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。

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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