From 2009.igem.org

(Difference between revisions)

Latest revision as of 18:39, 21 October 2009

Home of iGEMOSAKA wiki

NOTES

Calendar

July
M	T	W	T	F	S	S
		1	2	3	4	5
6	7	8	9	10	11	12
13	14	15	16	17	18	19
20	21	22	23	24	25	26
27	28	29	30	31

August
M	T	W	T	F	S	S
					1	2
3	4	5	6	7	8	9
10	11	12	13	14	15	16
17	18	19	20	21	22	23
24	25	26	27	28	29	30
31

September
M	T	W	T	F	S	S
	1	2	3	4	5	6
7	8	9	10	11	12	13
14	15	16	17	18	19	20
21	22	23	24	25	26	27
28	29	30

October
M	T	W	T	F	S	S
			1	2	3	4
5	6	7	8	9	10	11
12	13	14	15	16	17	18
19	20	21	22	23	24	25
26	27	28	29	30	31

November
M	T	W	T	F	S	S
						1
2	3	4	5	6	7	8
9	10	11	12	13	14	15
16	17	18	19	20	21	22
23	24	25	26	27	28	29
30

Registry Parts

All of Osaka's Parts and Devices is here

COLOR

K204033 mOrange

placI

K204041 placI+GFP

K204042 placI+YFP

K204043 placI+CFP

K204044 placI+RFP

K204048 placI+mOrange

pcI

K204058 pcI+YFP

K204059 pcI+CFP

K204062 pcI+mOrange

ptetR

K204052 ptetR+mOrange

Carotenoid

K204064 crtE+crtB

K204065 crtI+crtY

K204068 crtZ

K204076 placI+crtE+crtB

K204077 ptetR+crtE+crtB

K204091 placI+crtE+crtB+crtI+crtY

K204095 placI+crtE+crtB+crtZ

K204096 ptetR+crtE+crtB+crtZ

gradation

K204069 cI repressor

K204086 3O₆HSL receiver+RFP+cI repressor

SIGNAL (Quorum Sensing)

AI-1

K204047 LasI generater

C₄HSL

K204040 RhlI (generater)

K204031 RhlR (receiver)

K204702 RhlR+GFP

3OH,C₁₄ : 1-HSL

K204025 cinI (generater)

K204032 cinR (receiver)

3O₆HSL

K204051 LuxR (receiver)

K204700 LuxR+GFP

MOTILITY

K204018 ptetR+EspE

K204501 RBS+FliH

K204502 ptetR+FliH

K204503 pT7+FliH

K204504 C₄HSL receiver +FliH

Lab Note

Wet Lab

Week 1 : 8/2~8/8

Brainstorming , parts planning , prepared competent cell

ransformation and miniprep from MIT plate

week 2 : 8/9~8/15

start assembly parts (K204001 ~ K204006) , transformation and mini prep from MIT

Week 3 : 8/16~8/22

assembly parts (~K204015) , parts planning

Week 4 : 8/23~8/29

assembly parts (~K204021)

Week 5 : 8/30~9/5

assembly parts (~K204030)

Week 6 : 9/6~9/12

assembly parts (~K204046)

Week 7 : 9/13~9/19

assembly parts (~K204050)

Week 8 : 9/20~9/26

assembly parts (~K204056)

TEST : sensitivity of inducer and receptor

Sample : 3O₆HSL , C₄HSL , AI-1 , 3OH,C₁₄:1-HSL

Result : Not confirm the fluorescence...

Week 9 : 9/27~10/3

assembly parts (~K204066 , X1~X8), color (fluorescence) intensity check

Week 10 : 10/4~10/10

assembly parts (~K204078)

Lux receiver sensitivity test, color (fluorescence) intensity check

sequence check : generator and receiver

3O₆HSL , C₄HSL , AI-1 , 3OH,C₁₄:1-HSL

Get the parts from Team Chiba. Thanks a lot!!

Week 11 : 10/11~10/17

assembly parts (~K2040104)

sequence check : FliH etc…

send Osaka team parts to MIT

FINISH!

Finally we assembled about 120 parts!!

Dry Lab

Week 5 : 8/30~9/5

Planing model.

Week 6 : 9/6~9/12

Studying programing using Matlab.

Week 7 : 9/13~9/19

Making program in case of single coloney.

Week 8 : 9/20~9/26

Making program in case of more than two colonies

Making movie file

Searching paper written some parameter

Week 9 : 9/27~10/3

Blash up our program

Searching paper written some parameter

Week 10 : 10/4~10/10

Making program of color gradation

Week 11 : 10/11~10/17

Blash up our program

Atelie

Week 8 : 9/20~9/26

Making art works

Week 9 : 9/27~10/3

Making art works

Week 10 : 10/4~10/10

Making art works

Week 11 : 10/11~10/17

Making art works

@@ Line 175: / Line 175: @@
 <p>Week 11 : 10/11~10/17</p>
 <p>Making art works</p>
-<br>
    </div>
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-<div id="CollapsiblePanel4" class="CollapsiblePanel">
-  <div class="CollapsiblePanelTab" tabindex="0">
-    <h2>Protocols</h2></div>
-  <div class="CollapsiblePanelContent">
-    <h3>Sensor Experiment 1: Receivers vs AHL</h3>
-<h4>Day 1 - Preparation</h4>
-<p>1. From transformation plate, pick up a small quantity of cells from a colony using sterilized toothpick and streak on LA plate with the appropriate antibiotic resistance. Incubate at 37 degrees Celsius for 8+ hr.</p>
-<p>2. From the streak plate, choose a single-cell colony and pick up cells using sterilized toothpick, then inoculate 5ml LB culture medium to which the appropriate antibiotic resistance (50mg/l) has been added. Incubate overnight at 37 degrees Celsius in a shaking incubator.</p>
-<br>
-<h4>Day 2 - Measurement</h4>
-<p>1. Transfer 1ml of the overnight culture into 30ml fresh LB culture medium. Incubate in shaking incubator at 37 degrees.</p>
-<p>2. Measure OD of culture every hour or so, until OD reaches fixed value (culture at full growth).</p>
-<p>3. Transfer culture into 50ml ultracentrifuge tube and spin at 8000rpm for 5 minutes.</p>
-<p>4. Resuspend pellet with 30ml fresh LB culture.</p>
-<p>5. Incubate culture in shaking incubator at 30 degree Celcius for 10 minutes.</p>
-<p>6. Transfer 1ml of culture into microcentrifuge tube and put on ice. Immediately add AHL to rest of culture to attain desired concentration.
-(Note: Between the steps detailed below, samples should be kept on ice to minimize cellular activity.)</p>
-<p>7. Obtain OD600 of 2x diluted sample by transferring 500ul of sample into cuvette, adding 500ul MiliQ water and measuring the resulting OD.</p>
-<p>8. Ultracentrifuge remaining 500ul of sample at 13000rpm for 2min, discard supernatant, then resuspend pellet with 1ml MiliQ water.</p>
-<p>9. Ultracentrifuge sample again at 13000rpm for 2min, discard supernatant and resuspend with precisely 1ml MiliQ water (this results in 2x dilution).</p>
-<p>10. Measure GFP fluorescence with a fluorimeter at 488nm excitation, 513nm emission.</p>
-<p>11. Every hour, sample 1ml of culture and repeat steps 7-10 until fluorescence reaches steady state.</p>
-<br>
-<br>
-<h3>Sensor Experiment 2: Senders vs Receivers</h3>
-<h4>Day 1 - Preparation</h4>
-<p>1. From transformation plate, pick up a small quantity of cells from a colony using sterilized toothpick and streak on LA plate with the appropriate antibiotic resistance. Incubate at 37 degrees Celsius for 8+ hr.</p>
-<p>2. From the streak plate, choose a single-cell colony and pick up cells using sterilized toothpick, then inoculate 5ml LB culture medium to which the appropriate antibiotic resistance (50mg/l) has been added. Incubate overnight at 37 degrees Celsius in a shaking incubator.</p>
-<br>
-<h4>Day 2 - Mix & Culture</h4>
-<p>1. Prepare microcentrifuge tubes (3 samples for each sender-receiver pair). Add 400ul of LB-Amp culture medium to each microcentrifuge tube.</p>
-<p>2. Transfer 1ml of each overnight culture to separate microcentrifuge tubes and ultracentrifuge at 12000rpm for 1min. Discard supernatant and resuspend cells with 1ml LB-Amp. Repeat with remaining overnight culture if necessary to obtain the required amount of cell culture.</p>
-<p>3. For sender-receiver test: Add 50ul each of sender and receiver cultures to a microcentrifuge tube prepared in step 1. For the negative control (receiver only), add 100ul of receiver culture only.</p>
-<p>4. Incubate microcentrifuge tubes at 37 degrees Celsius overnight.</p>
-<br>
-<h4>Day 3 - Observation & Measurement</h4>
-<p>1. Wash cells by ultracentrifuging at 13000rpm for 2min, discarding supernatant and resuspending cells with MiliQ water. Repeat, and resuspend with precisely 1ml MiliQ water.</p>
-<p>2. For direct observation, bring cells suspended in water to darkened room and shine with UV Black Light. GFP fluorescence should be observable if sufficiently strong promoter activation has been achieved.</p>
-<p>3. Measure GFP fluorescence with a fluorimeter at 488nm excitation, 513nm emission.</p>
-  </div>
-  </div>
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Team:Osaka/NOTES

From 2009.igem.org

Latest revision as of 18:39, 21 October 2009

NOTES

Calendar

Registry Parts

All of Osaka's Parts and Devices is here

COLOR

SIGNAL (Quorum Sensing)

MOTILITY

Lab Note

Wet Lab

Get the parts from Team Chiba. Thanks a lot!!

Finally we assembled about 120 parts!!

Dry Lab

Atelie