Team:Chiba/Project/Experiments:Crosstalk
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===Details=== | ===Details=== | ||
+ | ====Method==== | ||
+ | 1.Crosstalk test | ||
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+ | To characterize quorum sensing crosstalk, constitutive AHL senders were mixed with constitutive receivers and measure fluorescence intensity. | ||
+ | |||
+ | #Transformed Senders into E.coli strains(XL10GOLD) and Receiver into E.coli strain(JW1908). | ||
+ | #Inoculated them independently in liquid media. Incubated at 37°C 12h | ||
+ | #Mixed them. | ||
+ | #Incubated at 30°C. | ||
+ | #Measured intensity of green fluorescence at regular time intervals. | ||
+ | |||
+ | [[Team:Chiba/protocol/phenotype/timedelay|more details...]] |
Revision as of 12:14, 29 October 2008
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Crosstalk
Design
Each species has their own LuxI-type proteins,which synthesize their specific autoinducers,AHLs.AHLs produced by different LuxI-type proteins differ only in the length of the acyl-chain moiety and substitution at position C-3.LuxR,which is original for Vibrio fischeri,is activated by its cognate autoinducer,3OC6HSL.However,LuxR is also activated by non-endogenous molecules,C4HSL,C6HSL,and 3OC12HSL.Activation by non-endogenous molecules requires a higher signal concentration(2).This results in slower activation of receivers,when AHL concentration is increasing.
異なる生物はそれぞれに異なるLuxIタイプのタンパク質を持ち、アシル鎖の長さ、あるいはC-3位の置換基が異なる種類のAHLを合成する。それぞれの生物種のLuxIタイプのタンパク質、それが合成する分子名は以下の表のようである。 (Fig.4).AHLを受け取り応答するLuxRタンパク質はVibrio fischeri由来であり、3OC6HSLに応答する。しかし、他種生物由来のAHLにも応答することが知られており、このとき、より高い濃度のAHLが必要となる(1),(2).AHLがゆっくり溜まっていく時、LuxRは3OC6HSLに対して最も早く応答し、他のAHLに対してはそれよりも遅く応答する。 (冨永)
Experiments
senders | receiver |
*[http://partsregistry.org/Part:BBa_K084007 plac+rbs+LasI] | *[http://partsregistry.org/Part:BBa_T9002 BBa_T9002 (Express GFP in response to AHL)] |
*[http://partsregistry.org/Part:BBa_K084008 plac+rbs+RhlI] | |
*[http://partsregistry.org/Part:BBa_K084012 plac+rbs+LuxI] |
Results&Discussion
Future plans
Details
Method
1.Crosstalk test
To characterize quorum sensing crosstalk, constitutive AHL senders were mixed with constitutive receivers and measure fluorescence intensity.
- Transformed Senders into E.coli strains(XL10GOLD) and Receiver into E.coli strain(JW1908).
- Inoculated them independently in liquid media. Incubated at 37°C 12h
- Mixed them.
- Incubated at 30°C.
- Measured intensity of green fluorescence at regular time intervals.