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Team:Osaka/NOTES
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NOTES
Calendar
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Registry Parts
Osaka team ALL PARTS hereParts & Devices
COLOR
K204033 mOrange
placIK204041 placI+GFP
K204042 placI+YFP
K204043 placI+CFP
K204044 placI+RFP
K204048 placI+mOrange
pcIK204058 pcI+YFP
K204059 pcI+CFP
K204062 pcI+mOrange
ptetRK204052 ptetR+mOrange
CarotenoidK204064 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
gradationK204069 cI repressor
K204086 3O6HSL receiver+RFP+cI repressor
SIGNAL (Quorum Sensing)
AI-1K204047 LasI generater
C4HSLK204040 RhlI (generater)
K204031 RhlR (receiver)
K204702 RhlR+GFP
K204025 cinI (generater)
K204032 cinR (receiver)
3O6HSLK204051 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 : 8/2~8/8Brainstorming , parts planning , prepared competent cell
transformation 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 the Chiba University Team. , Thanks chiba team
Week 11 : 10/11~10/17
assembly parts (~K2040104)
sequence check : FliH etc…
send Osaka team parts to MIT
FINISH!
Osaka team assembled about 120 parts!
Dry Lab
Week 5 : 8/30~9/5Planing 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 : 9/20~9/26Making 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
Protocols
Sensor Experiment 1: Receivers vs AHL
Day 1 - Preparation
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.
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.
Day 2 - Measurement
1. Transfer 1ml of the overnight culture into 30ml fresh LB culture medium. Incubate in shaking incubator at 37 degrees.
2. Measure OD of culture every hour or so, until OD reaches fixed value (culture at full growth).
3. Transfer culture into 50ml ultracentrifuge tube and spin at 8000rpm for 5 minutes.
4. Resuspend pellet with 30ml fresh LB culture.
5. Incubate culture in shaking incubator at 30 degree Celcius for 10 minutes.
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.)
7. Obtain OD600 of 2x diluted sample by transferring 500ul of sample into cuvette, adding 500ul MiliQ water and measuring the resulting OD.
8. Ultracentrifuge remaining 500ul of sample at 13000rpm for 2min, discard supernatant, then resuspend pellet with 1ml MiliQ water.
9. Ultracentrifuge sample again at 13000rpm for 2min, discard supernatant and resuspend with precisely 1ml MiliQ water (this results in 2x dilution).
10. Measure GFP fluorescence with a fluorimeter at 488nm excitation, 513nm emission.
11. Every hour, sample 1ml of culture and repeat steps 7-10 until fluorescence reaches steady state.
Sensor Experiment 2: Senders vs Receivers
Day 1 - Preparation
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.
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.
Day 2 - Mix & Culture
1. Prepare microcentrifuge tubes: 3 samples for each sender-receiver pair (sender-receiver test) or AHL concentration (receiver-only test). Add 400ul of LB-Amp culture medium to each microcentrifuge tube.
2. Add AHL to each microcentrifuge tube that will be used for receiver test.
3. 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.
4. For sender-receiver test: Add 50ul each of sender and receiver cultures to a microcentrifuge tube prepared in step 1. For receiver-only test: Add 100ul of receiver culture to a microcentrifuge tube that has been prepared in steps 1 & 2 (AHL added).
5. Incubate microcentrifuge tubes at 37 degrees Celsius overnight.
Day 3 - Observation & Measurement
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.
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.
3. Measure GFP fluorescence with a fluorimeter at 488nm excitation, 513nm emission.
