Team:Jilin China/Interlab
Interlab
Introduction
Over the past two years, iGEM has advanced the frontiers of science with the two biggest interlaboratory studies that have ever been done in the field of synthetic biology. The reason iGEM hold Interlab Study is "to obtain fluorescence data for three specific genetic devices expressing GFP from iGEM teams around the world”. We think it is a great chance for our team to make our own contribution to this project.
Devices we received
Test Device 1: J23101.B0034.E0040.B0015 in pSB1C3
Test Device 2: J23106.B0034.E0040.B0015 in pSB1C3
Test Device 3: J23117.B0034.E0040.B0015 in pSB1C3
Positive Control Device: I20270 in pSB1C3
Negative Control Device: R0040 in pSB1C3
Interlab
Protocols
1. Thaw competent cells on ice, dispose of unused competent cells.
2. Pipette 50µl of competent cells into 2ml tube (50µl in a 2ml tube per transformation).
3. Pipette 1µl of DNA into 2ml tube.
4. Pipette 1µl of control DNA into 2ml tube. Gently pipette up and down a few times.
5. Close 2ml tubes, incubate on ice for 30min
6. Heat shock tubes at 42°C for 1 min.
7. Incubate on ice for 5min.
8. Pipette 200µl SOC media to each transformation.
9. Incubate at 37°C for 2 hours.
10. Pipette each transformation on two petri plates for a 20µl and 200µl plating. Spread with sterilized spreader or glass beads immediately.
11. Incubate transformations overnight (14-18hr) at 37°C.
12. Pick single colonies.
13. Count colonies for control transformation: Count colonies on the 20μl control plate and calculate your competent cell efficiency. Competent cells should have an efficiency of 1.5x10^8 to 6x10^8 cfu/µg DNA.
14. Measure the total population using OD600. Measure the fluorescence of the instrument using a dilution series of this reference material to construct a standard curve.
Interlab
Results
Interlab
Results
Interlab
Results
Interlab
Results
Interlab
Results
Interlab
Results
Interlab
Conclusion
As our study showed, Device 2（J23106.B0034.E0040.B0015）was the strongest among the 3 promoters, which showed the highest GFP fluorescence. Device 2（J23106.B0034.E0040.B0015） was the highest, Device 1（J23101.B0034.E0040.B0015 ）was moderate, and Device 3（J23117.B0034.E0040.B0015 ）was the lowest.
Our own laboratory did not have the proper microplate reader, so we borrowed equipment from the Central Lab of General Biology in Jilin University. We could only use the microplate reader during the morning, so we had to start the experiment at one o’clock. We failed at the first time, and the result of our second try was not good as well. Our principal investigator - Dr. Hu encouraged us a lot and we finally got some good results finally. We were very grateful to Dr. Hu and those people who helped us a lot to operate the reader in Central Lab.
