Team:Botchan Lab Tokyo/InterLab




Inter Lab

Introduction "What is InterLab study?"

There are many labs researching in the field of synthetic biology. But the data in each lab, in each country or area are different sometimes since the protocols are different in each lab. These differences can sometimes affect the results because it will be difficult to compare the results done by different protocols even the aims of the experiments are the same. In this meaning, making a standard way of measurements is a big challenge but a very important thing for synthetic biology field. Interlab Study is aiming to improve the measurement tools based on the data collected from all over the world. This time, by using the standard protocol and the test devices provided from iGEM, teams conduct GFP fluorescence experiments to see and compare the results collected from teams all over the worlds.

 

Method and materials

Calibration protocols

1. OD600 Reference point

[Materials]
1 ml LUDOX-S40(provided in kit)
H2O
96 well plate (clear one)
Spectrophotometer (Molecular Devices Spectra Max 190)

[Method]
Added 100 µl LUDOX into wells A1,B1,C1,D1
Added 100 µl H2O into wells A2,B2,C2,D2
Measured absorbance 600 nm of all samples
Recorded the data

2. Fluorescein fluorescene standard curve

[Materials]
fluorescein (provided in kit)
10 ml 1xPBS
96 well plate(black)
Micro plate reader (Packard FluoroCoun)

Note : We used two type of the 96 well plates which are black one and clear one because we can't get the 96 well plate,black with flat, transparent/clear bottom.

[Method]
(1) Prepared the fluorescein stock solution
Prepared 2x fluorescein stock solution by resuspending fluorescein in 1 mL of 1xPBS.
Dilute the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50 µM

(2) Prepared the serial dilutions of fluorescein
Added 100 µl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12
Added 200 µl of fluorescein 1x stock solution into A1, B1, C1, D1.
Transfered 100 µl of fluorescein stock solution from A1 into A2.
Mixed A2 by pipetting up and down 3x and transfer 100 µl into A3.
Repeated this operation A3 to A4 until A11.
Repeated dilution series for rows B,C,D.
Measured fluorescene of all samples with micro plate reader.(Excitation 485 nm/Emission 530 nm)
Recorded the data.

Cell Measurement

[Materials]
Competent cells (Escherichia coli strain DH5α)
LB (Luria Bertani) media
Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 mg/mL)
Test tube(we used this for falcon tube in Day 2 operation.)
15 ml Falcon tube (covered in foil to block light)
Incubator at 37 °C
1.5 ml eppendorf tubes for sample storage
Ice bucket with ice
Pipettes
96 well plate, black and clear(two types)

Devices (from InterLab Measurement Kit):
⋅Positive control
⋅Negative control
⋅Test Device 1: J23101+I13504
⋅Test Device 2: J23106+I13504
⋅Test Device 3: J23117+I13504
⋅Test Device 4: J23101.BCD2.E0040.B0015
⋅Test Device 5: J23106.BCD2.E0040.B0015
⋅Test Device 6: J23117.BCD2.E0040.B0015

[Method]
Day 1: transform Escherichia coli DH5α

Day 2: Pick 2 colonies from each of plate and inoculate it on 5 mL LB medium + Chloramphenicol. Grow the cells overnight (16-18 hours) at 37 °C and 220 rpm.

Day3: Cell growth, sampling, and assay
Measured OD600 of the overnight cultures
Diluted the cultures to a target OD600 of 0.02 (see the volume of preloading culture and media in Excel (Dilution Calculation) Sheet_1) in 15 mL falcon tube (covered with foil to block light).
Incubated the cultures at 37 °C and 220 rpm.
Took 500 µL samples of the cultures at 0, 2, 4, and 6 hours of incubation (from 8 devices, in total 16 samples per point).
At the end of sampling point, measured Abs 600 and Fluorescence.
The measured fluorescence value was too strong and there were several error values, so we did the experiments from day2 again in following 2 days.

Day 4(Colony 1 only): Cell growth, sampling, and assay
Measured OD600 of the overnight cultures
Diluted the cultures to a target OD600 of 0.02 (see the volume of preloading culture and media in Excel (Dilution Calculation) Sheet_1) in 15 mL falcon tube (covered with foil to block light).
Incubated the cultures at 37 °C and 220 rpm.
Took 1000 µL samples of the cultures at 0, 2, 4, and 6 hours of incubation (from 8 devices).
(We forgot to place the samples on ice when we did this experiment of device 1.)
Pipeted 100 µl into well into wells A-D (96 well plates, one is transparent and the other is black.)
Measured Abs 600 and Fluorescence.

Day 5 (Colony 2): Cell growth, sampling, and assay
Measured OD600 of the overnight cultures
Diluted the cultures to a target OD600 of 0.02 (see the volume of preloading culture and media in Excel (Dilution Calculation) Sheet_1) in 15 mL falcon tube (covered with foil to block light).
Incubated the cultures at 37 °C and 220 rpm.
Took 1000 µL samples of the cultures at 0, 2, 4, and 6 hours of incubation (from 8 devices).
Pipeted 100 µl into well into wells E-F (96 well plates, one is transparent and the other is black.)
Measured Abs 600 and Fluorescence.

 

Result

The results of the OD600 measurement are on table 1 and figure 1. This data shows the growth of OD600 between 6 hours. In this data, OD600 of Device 1 and Device 4 are apparently low compared to the other devices. Other than these devices, the figure1 shows quite similar pattern of the growth.
The non-patterned result was observed in the measurement of the fluorescence. Many devices increased and decreased in each term.
Since the fluorescence's result affected to the absolute fluorescence, the absolute fluorescence also had a variation. But in all the devices, absolute fluorescence in 0h is the greatest. In positive control, negative control, device 1, device 2, and device 3, the absolute fluorescence decreased from 0h, but recovered a little in some point between 4h and 6h. In device 4, 5, and 6, the absolute fluorescence decreased over time. Device 3, device 5, device 6 shows little absolute fluorescence in all time. Especially in device 5 and 6, these 2 devices show little to no fluorescence.

Table1. OD600 measurement



Fig.1 OD600 measurement

 

Table2. Fluorescence Measurement



Fig.2 Relative Fluorescence Measurement

 

Table3. Measurement of Fl/Abs600



Fig. 3 Absolute Fluorescence Plot