The Fourth International InterLaboratory Measurement Study seeks to establish a reproducible plate reader-based GFP measurement protocol. Eight plasmids were transformed into competent DH5a E. coli cells and expressed for varying lengths of time. The optical density and fluorescence of each device was recorded over a 6 hour period. The major goal of this collective effort is to answer the following question: how close can the numbers be when fluorescence is measured all around the world?
Materials and Methods
In this Interlab study, the following 8 RBS devices were provided in the Kit Plate 7 in the 2017 Distribution Kit:
- Positive control
- Negative control
- Test Device 1:J23101.BCD2.E0040.B0015
- PTest Device 2: J23106.BCD2.E0040.B0015
- Test Device 3: J23117.BCD2.E0040.B0015
- Test Device 4: J23101+I13504
- Test Device 5: J23106+I13504
- Test Device 6: J23117+I13504
The transformation protocol was adapted from the iGEM protocol, which can be found on this link. All of the devices were transformed and cultured in LB (Luria Bertani) media containing 25mg/mL chloramphenicol. The following adjustments were made to the protocol:
- 2µL DNA were added to 50µL competent DH5a
- The incubation times on ice before and after the heat shock were 20 minutes and 2 minutes, respectively.
- The heat shock was performed for 90 seconds instead of 50 seconds.
- 800µL SOC broth were added to each transformation.
- After the one-hour shaking incubation, each transformation tube was centrifuged for 1 minute, 13,000 rpm. Afterwards, 700µL supernatant were discarded and the pellet was resuspended with the remaining 100µL before being plated onto LB agar plate.
On the next day, two colonies were picked from each plate and inoculated into two 5mL cultures, yielding 16 cultures in total. The cultures were subsequently incubated at 37 ºC and 220 rpm for 18 hours. Afterwards, the OD600 of the overnight cultures were measured using a spectrophotometer. The cultures were diluted to OD600 of 0.02 in 12mL LB medium and subsequently incubated at 37 ºC and 220 rpm. At time = 0, 2, 4, and 6 hours, 500µL sample from each culture was saved on ice, yielding 64 samples in the end. The dilution calculation can be found on the following link.
The 64 culture samples were transferred onto a clear, flat-bottomed 96-well plate according to the layout found on the following link. The OD600 absorption and fluorescence spectroscopy were measured using Synergy H1 Hybrid Multi-Mode Microplate Reader. The machine was previously calibrated using LUDOX. A fluorescein standard curve was also obtained under the same settings that were used to measure the culture samples.
The results of the Interlab Study were tabulated in the following link.