Team:Chiba/Project/Experiments:Receiver Crosstalk

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Revision as of 04:06, 30 October 2008

Receiver Cross-talk

Design

Fig. 1 Crosstalk

Table. 1 LuxR family gene Data modified from M.RIVAS et.al. Biol Res 38: 283-297, (2005) Table. 2 Comparison of the predicted Lux box. Data modified from A.Fekete et.al. Anal Bioanal Chem 387:455–467,(2007)

In this plan, we induce cross-talk of quorum sensing by changing receiver protein. It is known that receiver proteins can work with the stimuli of signaling molecule even from another species of bacteria (See Table. 1).⁽¹⁾⁽²⁾。
These endo-genieous combination of signaling molecules and receiver proteins causes the delay of response time and as a result the gene expression triggered by quorum sensing device is to be delayed.
For instance, receiver proteins, RhlR and LasR (from Pseudomonas aeruginosa) is stimulized by 3OC6HSL.

In addition, LuxR protein family activates the expression of gene which is located under a unique sequences of so-called Lux box promotor.(Table. 3)
Strictly speaking, the sequences of Lux box are dependent of the species of bacteria, however, they are quie similar so that we aim the gene expression of endo-geneous LuxR protein family with AHL.

Experiment

This experiment is for induction of cross-talk by changing LuxR protein family.
As AHL sender, the plasmid of Lux I protein from Vibrio fischeri was introduced and 3OC6HSL is synthesized by the sender.
As AHL Receiver, Receiver plasmid of LuxR protein family and the reporter plasmid of Lux promoter GFP were introduced. The AHL is produced by the AHL sender and stimulate the AHL receiver, triggering the GFP expression. We measured the fluorescence intensity of GFP using plate reader for investigation of activity of this cross-talk combination. The gene circuit used in this section is as follows.

Sender	Transcriptional regulator	Reporter
AHL sender[http://partsregistry.org/Part:BBa_S03623 (BBa_S03623) ]	LuxR	Plux-GFP[http://partsregistry.org/Part:BBa_J37032 (BBa_J37032)]
	RhlR[http://partsregistry.org/Part:BBa_K084004 (BBa_K084004)]
	LasR[http://partsregistry.org/Part:BBa_K084005 (BBa_K084005)]
	CinR[http://partsregistry.org/Part:BBa_K084006 (BBa_K084006)]

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 37c° 12h.
3. Inoculated again at 37c° upto about OD600=2.0
4. Washed them.
5. Mixed them (Sender:Receiver=1000μl:1000μl).
6. Incubated at 30c°.
7. Measured intensity of green fluorescence at regular time intervals.

Result & Discussion

Fig.4 Time Delay Test Sender:LuxI,Receiver:LuxR

Fig.5 Time Delay Test Sender:LuxI, Receiver:LasR

Fig.6 Time Delay Test Sender:LuxI, Receiver:RhlR

The diagrams are the time course change of fluorescence intensity of GFP expression indued by various combination of cross-talk using Lux protein family.

LuxR: high GFP expression level(Fig.4)
LasR: quite low GFP expression level(Fig.5, Fig.6)

As summary, in the cross-talk quorum sensing, the expression level is likely to decrease and then we could not recognize the delayed swith of GFP expression in these crosstalk combinatiom.

In this experiment, except LuxR, other R protein family proteins were tagged with LVA tag. Since LuxI->LasR->PLas and LuxI->RhlR->PRhl curcuit provides low expression level[http://www3.interscience.wiley.com/journal/119124142/abstract (1)], this experimental result is from the LVA tag. The experiment usig combination of Sender:LuxI (no LAV tag), Receiver:LuxR,LasR,RhlR (no LAV tag) is furhter necessary to discuss on the current result.

Demo ~Receivers~

Breif explanation on Methods:
1. LB pre-cultured Sender(BBa_S03623(JW1908)) was mixed with LB-agar to produce sender containing bacterial plate.
2. Receiver colony was transfered to a nitrocellulose filter and placed on a Sender(BBa_S03623(JW1908)) containing bacterial plate.
3. AHL produced by sender is diffused in the LB-agar plate and the receivers on the nitrocellulose filter synthesize GFP when they are stimulated by a certain amount of AHL.
4. Plates cultured at 37°C were exposed to UV (312nm) light once every 30 minutes to observe GFP fluorescence.

The receivers are:

• AiiA synthesizing receiver.
  
• receiver which has different copy number.
  
• Cross-talk receiver.
  

Results

--->more about Demo experiments detail

Reference

1. [http://www3.interscience.wiley.com/journal/119124142/abstract M.K Winson et al.:Construction and analysis of luxCDABE-based plasmid sensors　for investigating N-acyl homoserine lactone-mediated quorum sensing.FEMS Microbiology Letters 163 (1998) 185-192]
2. [http://partsregistry.org/Part:BBa_F2620:Specificity BBa_F2620:Specificity]

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