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	#Dilute receiver cells and spread on agar plate, and spot sender 1μL. Incubate at 37℃ 12hour		#Dilute receiver cells and spread on agar plate, and spot sender 1μL. Incubate at 37℃ 12hour
	#Put plate 4℃		#Put plate 4℃
-	#Check GFP expression every 1hour.	+	#Check GFP expression every 1hour.
-		+
-	*実験方法は？by とよたろ	+
	====Result====		====Result====

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Revision as of 00:09, 27 October 2007

Introduction | Project Design ( 1.Sticky Hands | 2.Communication | 3.Size Control ) | Making Marimos | Our Goal || Team Members | メンバ連絡簿

Size Control

Our Aim

Since produced AHL diffuses all around the bacteria culture, all receivers can ultimately aggregate to one huge marimo with their sticky hands. This final state is not the case of real marimos. Thus our idea for controlling the size of Bacteria Marimos is based on the high performance quorum sensing or AHL-diffusing inhibition.

1. Raise the AHL productivity of Sender
2. Increase the AHL sensitivity of Receiver
3. Use the AHL degrading enzyme aiiA to localize AHL

1.Improving Sender

Design

AHL is synthesized from methionine by the enzyme MetK and LuxI.
We tested in the hope of combined overexpression of these 2 genes will enhance the sender capacity.

Experiment

Sender

Ptet-LuxI

• Synthesize AHL constantly

metK Sender

• Synthesize AHL and express metK constantly

Receiver

Method(Fig3.)

1. Inoculated sender, MetK sender, and receiver in each liquid medias. Incubated at 37℃ 12h.
2. Checked OD. Dispensed receiver in each tube equally. Spin downed senders and resuspended with fresh liquid media.
3. Diluted senders to adjust cell population (5x10⁸,5x10⁷,......, 5x10²).
4. Mix receivers and senders.
5. Incubated for 1h to 3h at room tempature
6. Spindowned and UV checked.

Result

• No difference was seen.

Between Methionine added or not.
Between metK is expressed or not.

• 何と何の間の違いが見られないのかな？byとよたろ
  

Discussion

• No efficiency was observed.
• Our Assumption:S-adenosyl methionine was synthesized enough or consumed in other path way.
• Sequencing riquired.

2.Improving Receiver

Design

Collins et.al. described the hyper-sensitive variants of luxR to AHL.(Collins, C. H., Arnold, F. H. & Leadbetter, J. R. Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones. Mol. Microbiol. 55, 712–723 (2005))
We created two types of sensitive luxR mutants.
Because there is no data shows 2point(I45F,S116A) mutated luxR．We expected something new data of mutation will be found. Also we want to obtain sensitive(not hyper sensitive) receiver.

Experiment

Sensitive luxR mutants assessed by follow method.

1.Inoculate Receiver(wild type luxR/BW25113), mutated Receiver(1point mutation/BW25113) in liquid media 37℃ 12hour.
2.Despence them equally.
3.Add AHL final concentration(1μM,100nM,10nM,5nM,1nM,0.1nM)
4.1hour at room temperature
5.Spindown and UV checked.

Result

• Both wild type and 1point mutation luxR express GFP under 5nM AHL concentration.

• 2point mutation(I45F,S116A) luxR Receiver express GFP without AHL.

Discussion

This table is quoted from paper.
In the paper, DH5α strain was used.
But wild type luxR in our study expressed GFP under 5nM AHL concentration.

Assumption:There is some difference in strains.

3.Localizing AHL

Design1

AiiA expression is regulated by pLac, which makes it express constantly to inactivate AHL.

Experiment

1. Inoculate E.coli carries this aiiA receiver plasmid in Liquid Media.
2. At the same time, inoculate AHL sender E.coli in Liquid Media.
3. Dilute receiver cells and spread on agar plate, and spot sender 1μL. Incubate at 37℃ 12hour
4. Check plate.(GFP expressed?)

Result

GFP expression was not detected on the plate of aiiA receiver.

Discussion

Excessive amounts of　aiiA is expressed to generate concentration gradient. We expected aiiA express more moderate to make it.

Design2

This gene circuit is expected so that aiiA is synthesized only when the receiver senses AHL signal. If so, the whole Bacteria Marimo becomes AHL quencher. In fact, the bacteria inside of the Marimo degrades AHL and the AHL diffusion outside of Marimo is inhibited.

Experiment

1. Inoculate E.coli carries this aiiA receiver plasmid in Liquid Media.
2. At the same time, inoculate AHL sender E.coli in Liquid Media.
3. Dilute receiver cells and spread on agar plate, and spot sender 1μL. Incubate at 37℃ 12hour
4. Put plate 4℃
5. Check GFP expression every 1hour.

Result

• When we compared the GFP expression with the test tube of BBa_T9002, the bacteria with the consructed gene circuit did not fluoresce immediately after incubation, however fluoresced in 18~20 hour.
  
• しばらくすると、ってどのくらいのタイムスケールですか？by とよたろ

Discussion

• GFP expression means plux is activated. So we could make moderate aiiA receiver.
• But we need more good scheme.

Design3

Above picture describes aiiA is regulated inverted lux promoter with CI inverter．

• High AHL concentration: no aiiA expression, no AHL degrade.
• Low AHL concentration: aiiA expressed, AHL degraded.

図のようにインバーターをかませた．高い濃度ではaiiAが発現しないためAHLは分解されない．AHLが低い濃度ではaiiAが発現しAHLを分解する．

よってマリモの中心付近ではAHLを分解せず，外側へ行くと分解される．

Experiment

We could not finish assembling this part. It　will be our future work. このパーツを作ろうとしたが、inverterを組み合わせた時点で終わってしまった。

Subpart:BBa_S03840