Team:Chiba/Project/Experiments:Signal Molecule Quencher

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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

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.


Notes 

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 37C)
  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.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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