Team:Chiba/Project/Experiments:Signal Molecule Quencher

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Revision as of 01:19, 30 October 2008

AiiA Receiver

Design

Design

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)]

AiiA Receiver

non-AiiA Receiver

Method

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

Result & Discussion

Results and discussion 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

[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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-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。
+Results and discussion
-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。
+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.
-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。
+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.
-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===