Team:UESTC-China/test-InterLab
Background
In order to repeat measurements in different labs, iGEM has proposed Interlab studies. The aim is to improve the measurement tools available to both the iGEM community and the synthetic biology community as a whole. This is an optional and voluntary exercise for all teams. This is also an optional activity for one the Bronze Medal Requirements this year. This year’s gaol was to measure the GFP fluorescence in a plate reader to test some RBS devices (BCDs) that are intended to make gene expression more precise and reliable.
Overview
Reliable and repeatable measurement is a key component to all engineering disciplines. So one of the big challenges in synthetic biology measurement is that fluorescence data usually cannot be compared because it is reported in different units or because different groups process data in different ways. Often working around this by doing some sort of “relative expression” comparison; however, being unable to directly compare measurements makes it harder to debug engineered biological constructs, harder to effectively share constructs between labs, and harder even to just interpret your experimental controls.
Thanks to iGEM’s interLab protocol for measuring GFP fluorescence, we have a chance to participate in the Fourth International InterLaboratory Measurement Study in synthetic biology. Our team also participated in the measurement study last year. Through learning from them, we have confident in doing this better this year. With minor difficulties we managed to complete the experiments and submit our results.
Devices
The 2017 iGEM teams were provided with six test devices, including two controls, a positive and negative control
Kit Plate 7 InterLab Part Locations
Positive Control (BBa_I20270)
Negative Control (BBa_R0040)
Test Device 1 (BBa_J364000)
Test Device 2 (BBa_J364001)
Test Device 3 (BBa_J364002)
Test Device 4 (BBa_J364003)
Test Device 5 (BBa_J364004)
Test Device 6 (BBa_J364005)
Each of the test devices consisted of promotors J23101, J23106, J23117, J23101, J23106, J23117 respectively. The rest of the test devices was identical consisting of the RBS, the terminator and the GFP (E0040).
Each device is in the pSB1C3 plasmid backbone, so transform all of the devices onto plates containing chloramphenicol.
Materials and Methods
Thermo MULTISKAN GO plate reader
HITACHI F-4600 Fluorescence spectrophotometer
Competent cells (Escherichia coli strain DH5α)
We do our interlab study depends on Plate Reader Protocol
Calibration Protocols
1.OD600 Reference point
1ml LUDOX (provided in kit)
H 2 0 (provided by team)
96 well plate, black with flat bottom preferred (provided by team)
1. Add 100 μl LUDOX into wells A1, A2, A3, A4
2. Add 100 μl H2O into wells B1, B2, B3, B4
3. Measure absorbance 600 nm of all samples in plate reader
4. Report and import data into Excel
2. Protocol fluorescein fluorescence standard curve
fluorescein (provided in kit)
10ml 1xPBS (phosphate buffered saline; provided by team)
96 well plate, black with flat bottom preferred (provided by team))
◻◻ Prepare the fluorescein stock solution
◻◻Prepare the serial dilutions of fluorescein
1. Spin down FITC stock tube to make sure pellet is at the bottom of tube.
2. Prepare 2x FITC stock solution (500 μM) by resuspending FITC in 1 mL of 1xPBS
3. Incubate the solution at 42°C for 4 hours.Check if the FITC is properly dissolved by pipetting up and down
4. Dilute the 2x FITC stock solution in half with 1xPBS to make a 1x FITC solution and resulting concentration of FITC stock solution 250 μM.
5. Prepare 12 2.0 mL tubes
6. Add 1 mL of PBS into tubes 2-11
7. Add 2.0 mL of 1x PBS into tube 12
8. Add 2.0 mL of FITC 1x stock solution into tube 1
9. Transfer 1.0 ml of FITC stock solution tube 1 into tube 2
10. Mix tube 2 by pipetting up and down 3x and transfer 1 mL into tube 3…
11. Mix tube 3 by pipetting up and down 3x and transfer 1 mL into tube 4…
12. Mix tube 4 by pipetting up and down 3x and transfer 1 mL into tube 5…
13. Mix tube 5 by pipetting up and down 3x and transfer 1 mL into tube 6…
14. Mix tube 6 by pipetting up and down 3x and transfer 1 mL into tube 7…
15. Mix tube 7 by pipetting up and down 3x and transfer 1 mL into tube 8…
16. Mix tube 8 by pipetting up and down 3x and transfer 1 mL into tube 9…
17. Mix tube 9 by pipetting up and down 3x and transfer 1 mL into tube 10…
18. Mix tube 10 by pipetting up and down 3x and transfer 1 mL into tube 11…
19. Mix tube 11 by pipetting up and down 3x and and transfer 1 mL into liquid waste.
20. Put the 12 tubes in dark
Cell measurement protocol
Competent cells (Escherichia coli strain DH5α)
LB (Luria Bertani) media
Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 ug/mL)
50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)
Incubator at 37°C
1.5 ml eppendorf tubes for sample storage
Ice bucket with ice
Pipettes
1. Wash the cuvette with UP water
2. Add 1 mL of UP water into the cuvette, measure
3. Wash the cuvette with 1x PBSs
4. Measure the sample one by one, each measure 4 times and wash the cuvette with 1xPBS between two samples.
5. The order of the measurement is tube 12, tube 1, tube 11, tube 10, tube 9, tube 8,tube 7, tube 6, tube 5, tube 4, tube 3, tube 2
6. Record the data
Results and Discussion
