InterLab 2016 is the third, and largest, interlaboratory study ever conducted in synthetic biology. Data from the two previous InterLab studies is summarised in a paper by Beal et al. 2016. The study aims to standardize the measurement of fluorescence in labs around the world by testing replicability of data. This time it is done by following the same protocols designed for a plate reader and a flow cytometer that will produce data in same units everywhere so it can be compared and analysed. Team Newcastle decided to test both protocols to measure the fluorescence.
InterLab Measurement Kit
This year teams were provided with the InterLab Measurement Kit which contained 7 tubes (5 with plasmid DNA and 2 with standard reagents):
Plasmid DNA (100 pg/uL in 10 uL of Buffer EB)
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
FITC Standard: one tube with dried down FITC for creating a FITC standard
LUDOX: one tube with 30% colloidal silica suspended in 1 mL of water
E. cloni® 10G Chemically Competent Cells were used for the transformation of bacteria. You can find the protocol we use for our transformations in our protocol library.
Transformed cells were plated on Petri dishes and grown in liquid cultures with appropriate antibiotic. They were prepared by following an official 2016 iGEM InterLab Measurement Study Plate Reader protocol and growing cultures in 10 ml LB and 10 µl of 1000x chloramphenicol mixed medium.
2016 iGEM InterLab Measurement Study Plate Reader Protocol
First, fluorescence was measured with FLUOstar OMEGA Microplate Reader provided by BMG Labtech while following updated 2016 iGEM InterLab Measurement Study Plate Reader Protocol that can be found here.
While following cell measurement section of the protocol above, the target OD600 achieved after the first measurement with an Eppendorf Biophotometer and dilution according to the calculations in the sheet provided was 0.03 in all of the samples. The data which was obtained from the plate reader measurements can be found below.
2016 iGEM InterLab Measurement Study Flow Cytometry Protocol
2016 iGEM InterLab Measurement Study Flow Cytometry Protocol was followed to measure fluorescence by flow cytometry. FACSCanto II flow cytometer configured for measurement of GFP and SpheroTech Rainbow Calibration Particles RCP-30-5A were used as recommended in the protocol. The team were guided all the way by the members of staff working at the Flow Cytometry Core Facility, Newcastle University to make sure that the machine was used properly and measurements obtained are right.
However, a few limitations might have affected the results. First of all, the protocol page did not have clear instructions how to prepare the cultures so they were diluted 2:3, and then 1:100 times to be measured in the flow cytometer. It would be useful to have a full flow cytometry protocol to avoid differences in dilutions. Also, the geometric mean could not be obtained for the negative control and devices that had no GFP expression due to them having a negative value. Thus, other types of data could be collected to get more accurate and consistent data.
Both Plate Reader and Flow Cytometry results indicate that Device 1 is the closest to positive control which means that it has the strongest promoter and the best RBS. The other two devices produce results which are close to negative control data. This indicates that they have very week or non-functioning promoters and RBS.
Our results can be found here