Team:Chiba/Project/Experiments:Signal Molecule Quencher

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===Design===
===Design===
'''AiiA added to Lux reporter'''
'''AiiA added to Lux reporter'''
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[[Image:Chiba project design.jpg|frame|left|'''Fig. 1'''       Attenuating the cellular communication]]
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[[Image:Chiba project design.jpg|frame|left|'''Fig. 1　Attenuating the cellular communication''' ]]
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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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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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[[Image:Aiia reaction chiba.gif|frame|center|'''Fig. 2　AiiA Reaction''' ]]
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'''Detailed Condition'''
'''Detailed Condition'''
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Sender; LuxI plasmid transformed into E.coli strains (JW1908).<br>
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Sender; LuxI plasmid transformed into ''E. coli'' strains (JW1908).<br>
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Receiver; LuxR-gfp plasmid transformed into E.coli strains (JW1908).<br>
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Receiver; LuxR-gfp plasmid transformed into ''E. coli'' strains (JW1908).<br>
Culture/ Cndn.
Culture/ Cndn.
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2.Inoculated into flesh media, shaken until cell density hit 2.0 in OD600<br>
2.Inoculated into flesh media, shaken until cell density hit 2.0 in OD600<br>
3.Washed the cell and re-suspended. Cell density checked.<br>
3.Washed the cell and re-suspended. Cell density checked.<br>
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4.Mixed Sender and Receiver (Sender/Receiver 1000μl/1000μl).<br>
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4.Mixed Sender and Receiver (Sender/Receiver 1000&mu;L/1000&mu;L).<br>
5.Incubated at 30°C.<br>
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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6.Time-chased the fluorescence (485nm (excitation) and 527nm (emission)) by gfp.<br>
{| class="tbl" |
{| class="tbl" |
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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).
#Inoculated them independently in liquid media. Incubated at 37&deg;C 12h.
#Inoculated them independently in liquid media. Incubated at 37&deg;C 12h.
#Inoculated again at 37&deg;C upto about OD600=2.0
#Inoculated again at 37&deg;C upto about OD600=2.0
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===Result & Discussion===
===Result & Discussion===
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[[Image:AiiA-Receiver-result Chiba.gif|frame|left|'''Fig. 3''' Time Delay Test +AiiA Receiver vs. -AiiA Receiver]]
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[[Image:AiiA-Receiver-result Chiba.gif|frame|left|'''Fig. 3　Time Delay Test +AiiA Receiver vs. -AiiA Receiver''' ]]
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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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