InterLab
Introduction
Reliable and repeatable measurement is a key component to all engineering disciplines. The same holds true for synthetic biology, which has also been called engineering biology. However, the ability to repeat measurements in different labs has been difficult.
This year is fourth InterLab study that organized by the iGEM Organization Measurement Committee. It aims to establish a GFP measurement protocol based on engineering principles that anyone with a plate reader can use in their lab and to improve measurement tools and methods in laboratory work.
Experiment
The whole work flow:
This year is fourth InterLab study that organized by the iGEM Organization Measurement Committee. It aims to establish a GFP measurement protocol based on engineering principles that anyone with a plate reader can use in their lab and to improve measurement tools and methods in laboratory work.
The first day: Parts collection and transformation
Agents and instruments that we used:
1. InterLab Measurement Kit supplied by iGEM
2. Escherichia coli K-12 DH5α competent cells provided by ourselves
3. Water bath
4. Shake cultivation
5. Chloramphenicol as antibiotic
6. LB medium (liquid and agar plate)
1. We transformed 8 parts listed in table 1 from the plate of InterLab Measurement Kit according to the given transformation protocol using Escherichia coli K-12 DH5α competent cells as required.
|
Part name |
Part ID |
Location |
|
Positive Control |
BBa_I20270 |
21B |
|
Negative Control |
BBa_R0040 |
21D |
|
Test Device 1 |
BBa_J364000 |
21F |
|
Test Device 2 |
BBa_J364001 |
21H |
|
Test Device 3 |
BBa_J364002 |
21J |
|
Test Device 4 |
BBa_J364003 |
21L |
|
Test Device 5 |
BBa_J364004 |
21N |
|
Test Device 6 |
BBa_J364005 |
21P |
The second day: Colonies picking and cell growth
Agents and instruments that we used:
1. Shake cultivation
2. Chloramphenicol as antibiotic
3. LB medium (liquid and agar plate)
4. 8 plates with colonies
Procedure:
1. We pick 2 colonies from each of plate and inoculate it on 5-10 mL LB medium + Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm.
The third day: Calibration, cell growth, sampling, and assay
Part I: Measurement of reference point of OD 600
Agents and instruments that we used:
1. InterLab Measurement Kit supplied by iGEM
2. Microplate Reader: PerkinElmer Enspire 2300
3. H20
4. 96 well plate, black with flat, transparent.
Procedure:
1. Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)
2. Add 100 μl of H2O into wells A2, B2, C2, D2 (or 1 mL H2O into cuvette)
3. Measure absorbance 600 nm of all samples in all standard measurement modes in
Instrument.
4. Record the data in the table below or in your notebook
5 Import data into Excel (OD600 reference point tab)
Results of part I
|
|
LUDOX-HS40 |
H2O |
|
Replicate 1 |
0.047 |
0.037 |
|
Replicate 2 |
0.045 |
0.037 |
|
Replicate 3 |
0.047 |
0.037 |
|
Replicate 4 |
0.046 |
0.037 |
|
Arith. Mean |
0.04625 |
0.037 |
|
Corrected Abs600 |
0.00925 |
|
|
Reference OD600 |
0.0425 |
|
|
OD600/Abs600 |
4.594594595 |
|
Part II: Making the standard curve of fluorescein fluorescence
Agents and instruments that we used:
1. fluorescein (provided in kit)
2. 10ml 1xPBS
3. 96 well plate, black with flat, transparent.
1. Prepare the fluorescein stock solution as the protocol provided by iGEM.
2. Prepare the serial dilutions of fluorescein as the protocol. 3. Repeat dilution series for rows B, C, D
4. Measure fluorescence of all samples in all standard measurement modes in instrument
5. Record the data in your notebook
6. Import data into Excel (fluorescein standard curve tab)
Results of part II
Part III: Cell measurement
Agents and instruments that we used:
1. Microplate Reader: PerkinElmer Enspire 2300
2. Devices cultured in the first two days
3. 96 well plate, black with flat, transparent.
4. 50 mL falcon tube
Procedure:
1. Set the instrument to read OD600 (as OD calibration setting)
2. Measure OD600 of the overnight cultures
3. Record data
4. Import data into Excel
5. Dilute the cultures to a target OD600 of 0.02 in 12 ml LB medium + Chloramphenicol
in 50 mL falcon tube (amber, or covered with foil to block light).
6. Incubate the cultures at 37°C and 220 rpm.
8. Take 500 μL samples of the cultures at 0, 2, 4, and 6 hours of incubation. (At each time
point, we took a sample from each of the 8 devices, two colonies per device, for a total of 16 samples per time point)
9. Place samples on ice.
10. Measure samples at the end of sampling point you need to (OD and Fl measurement).
11. Record data in the notebook
12. Import data into Excel.
Results of part III
