Team:Osaka/NOTES

From 2009.igem.org

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<p>assembly parts (~K204030)</p>
<p>assembly parts (~K204030)</p>
<br>
<br>
-
</p>Week 6 : 9/6~9/12</p>
+
<p>Week 6 : 9/6~9/12</p>
<p>assembly parts (~K204046)</p>
<p>assembly parts (~K204046)</p>
<br>
<br>
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<p>sequence check : generator and receiver</p>
<p>sequence check : generator and receiver</p>
<p>3O<sub>6</sub>HSL , C<sub>4</sub>HSL , AI-1 , 3OH,C<sub>14</sub>:1-HSL</p>
<p>3O<sub>6</sub>HSL , C<sub>4</sub>HSL , AI-1 , 3OH,C<sub>14</sub>:1-HSL</p>
-
Get the parts from Team Chiba. Thanks a lot!!
+
<h4 style="text-indent:2em">Get the parts from Team Chiba. Thanks a lot!!</h4>
<br>
<br>
 +
<p>Week 11 : 10/11~10/17</p>
 +
<p>assembly parts (~K2040104)</p>
 +
<p>sequence check : FliH etc…</p>
 +
<p>send Osaka team parts to MIT</p>
 +
<p>FINISH!</p>
 +
<h4 style="text-indent:2em">Finally we assembled about 120 parts!!</h4>
<br>
<br>
-
Week 11 : 10/11~10/17<br>
+
<h3>Dry Lab</h3>
-
assembly parts (~K2040104)<br>
+
<p>Week 5 : 8/30~9/5</p>
-
sequence check : FliH etc…<br>
+
<p>Planing model.</p>
-
send Osaka team parts to MIT<br>
+
-
FINISH!<br>
+
-
<b>Osaka team assembled about 120 parts!</b><br>
+
-
 
+
<br>
<br>
-
<h4><p>Dry Lab</p></h4>
+
<p>Week 6 : 9/6~9/12</p>
-
Week 5 : 8/30~9/5<br>
+
<p>Studying programing using Matlab. </p>
-
Planing model.<br>
+
-
 
+
<br>
<br>
-
Week 6 : 9/6~9/12<br>
+
<p>Week 7 : 9/13~9/19</p>
-
Studying programing using Matlab. <br>
+
<p>Making program in case of single coloney.</p>
-
 
+
<br>
<br>
-
Week 7 : 9/13~9/19<br>
+
<p>Week 8 : 9/20~9/26</p>
-
Making program in case of single coloney.<br>
+
<p>Making program in case of more than two colonies</p>
-
 
+
<p>Making movie file</p>
 +
<p>Searching paper written some parameter</p>
<br>
<br>
-
Week 8 : 9/20~9/26<br>
+
<p>Week 9 : 9/27~10/3</p>
-
Making program in case of more than two colonies<br>
+
<p>Blash up our program </p>
-
Making movie file<br>
+
<p>Searching paper written some parameter</p>
-
Searching paper written some parameter<br>
+
-
 
+
<br>
<br>
-
Week 9 : 9/27~10/3<br>
+
<p>Week 10 : 10/4~10/10</p>
-
Blash up our program <br>
+
<p>Making program of color gradation</p>
-
Searching paper written some parameter<br>
+
-
 
+
<br>
<br>
-
Week 10 : 10/4~10/10<br>
+
<p>Week 11 : 10/11~10/17</p>
-
Making program of color gradation<br>
+
<p>Blash up our program</p>
-
 
+
<br>
<br>
-
Week 11 : 10/11~10/17<br>
+
<h3>Atelie</h3>
-
Blash up our program<br>
+
<p>Week 8 : 9/20~9/26</p>
 +
<p>Making art works</p>
<br>
<br>
-
<h4><p>Atelie</p></h4>
+
<p>Week 9 : 9/27~10/3</p>
-
Week 8 : 9/20~9/26<br>
+
<p>Making art works</p>
-
Making art works<br>
+
-
 
+
<br>
<br>
-
Week 9 : 9/27~10/3<br>
+
<p>Week 10 : 10/4~10/10</p>
-
Making art works<br>
+
<p>Making art works</p>
-
 
+
<br>
<br>
-
Week 10 : 10/4~10/10<br>
+
<p>Week 11 : 10/11~10/17</p>
-
Making art works<br>
+
<p>Making art works</p>
-
 
+
-
<br>
+
-
Week 11 : 10/11~10/17<br>
+
-
Making art works<br>
+
-
 
+
-
 
+
   </div>  
   </div>  
   </div>  
   </div>  
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    <h2>Protocols</h2></div>
 
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  <div class="CollapsiblePanelContent">
 
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    <h3>Sensor Experiment 1: Receivers vs AHL<h3>
 
-
 
-
<p>Day 1 - Preparation</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.<br>
 
-
 
-
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.<br>
 
-
 
-
 
-
<br>
 
-
Day 2 - Measurement<br>
 
-
1. Transfer 1ml of the overnight culture into 30ml fresh LB culture medium. Incubate in shaking incubator at 37 degrees.<br>
 
-
<br>
 
-
2. Measure OD of culture every hour or so, until OD reaches fixed value (culture at full growth).<br>
 
-
<br>
 
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3. Transfer culture into 50ml ultracentrifuge tube and spin at 8000rpm for 5 minutes.<br>
 
-
<br>
 
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4. Resuspend pellet with 30ml fresh LB culture.<br>
 
-
<br>
 
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5. Incubate culture in shaking incubator at 30 degree Celcius for 10 minutes.<br>
 
-
<br>
 
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6. Transfer 1ml of culture into microcentrifuge tube and put on ice. Immediately add AHL to rest of culture to attain desired concentration.<br>
 
-
<br>
 
-
(Note: Between the steps detailed below, samples should be kept on ice to minimize cellular activity.)<br>
 
-
<br>
 
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7. Obtain OD600 of 2x diluted sample by transferring 500ul of sample into cuvette, adding 500ul MiliQ water and measuring the resulting OD.<br>
 
-
<br>
 
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8. Ultracentrifuge remaining 500ul of sample at 13000rpm for 2min, discard supernatant, then resuspend pellet with 1ml MiliQ water.<br>
 
-
<br>
 
-
9. Ultracentrifuge sample again at 13000rpm for 2min, discard supernatant and resuspend with precisely 1ml MiliQ water (this results in 2x dilution).<br>
 
-
<br>
 
-
10. Measure GFP fluorescence with a fluorimeter at 488nm excitation, 513nm emission.<br>
 
-
<br>
 
-
11. Every hour, sample 1ml of culture and repeat steps 7-10 until fluorescence reaches steady state.<br>
 
-
<br>
 
-
<br>
 
-
 
-
Sensor Experiment 2: Senders vs Receivers<br>
 
-
 
-
Day 1 - Preparation<br>
 
-
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.<br>
 
-
<br>
 
-
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.<br>
 
-
<br>
 
-
 
-
Day 2 - Mix & Culture<br>
 
-
1. Prepare microcentrifuge tubes (3 samples for each sender-receiver pair). Add 400ul of LB-Amp culture medium to each microcentrifuge tube.<br>
 
-
<br>
 
-
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.<br>
 
-
<br>
 
-
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.<br>
 
-
<br>
 
-
4. Incubate microcentrifuge tubes at 37 degrees Celsius overnight.<br>
 
-
<br>
 
-
 
-
Day 3 - Observation & Measurement<br>
 
-
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.<br>
 
-
<br>
 
-
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.<br>
 
-
<br>
 
-
3. Measure GFP fluorescence with a fluorimeter at 488nm excitation, 513nm emission.<br>
 
-
<br>
 
-
<br></p><br>
 
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-
 
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Latest revision as of 18:39, 21 October 2009

Home of iGEMOSAKA wiki

NOTES

Calendar

July
MTWTFSS
    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
MTWTFSS
          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
MTWTFSS
  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
MTWTFSS
      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
MTWTFSS
            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


Retrieved from "http://2009.igem.org/Team:Osaka/NOTES"