Team:Newcastle/InterLab
Interlab
Introduction
InterLab study 2016 is the largest interlaboratory study in synthetic biology. It 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 10uL of Buffer EB) o Test Device 1: J23101.B0034.E0040.B0015 in pSB1C3 o Test Device 2: J23106.B0034.E0040.B0015 in pSB1C3 o Test Device 3: J23117.B0034.E0040.B0015 in pSB1C3 o Positive Control Device: I20270 in pSB1C3 o 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 1mL of water
Protocols
E. cloni® 10G Chemically Competent Cells were used for the transformation of bacteria. The protocol followed can be found here: http://www.lucigen.com/docs/manuals/MA010-Ecloni-10G-Chem-Comp.pdf
Transformed cells were plated on Petri dishes and grown in liquid cultures. They were prepared by following an official 2016 iGEM InterLab Measurement Study Plate Reader protocol and growing cultures in 10 ml LB and 10µl 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: https://static.igem.org/mediawiki/2016/c/c5/InterLab_iGEM2016_Plate_Reader_Protocol_Updated_July.pdf
While following cell measurement section of the protocol above, the target OD600 achieved after the first measurement with (insert brand) spectrophotometer 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 (https://2016.igem.org/Tracks/Measurement/Interlab_study/Flow_Cytometry_Overview) 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 of the 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, 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 type of data could be collected to get more accurate and consistent data.
