All iGEM 2016 were encouraged to be a part of InterLab Measurement Study. Participation was voluntary for all teams. This study is establishing a baseline for replicability of fluorescence measurements and identified likely key sources of error.
One of the biggest challenges in synthetic biology is that measurements of fluorescence usually cannot be compared because they are reported in different units or because different groups process data in different ways. This study focused on one of the most widely used tools in genetic engineering: constitutive expression of fluorescent protein from an engineered plasmid. Teams, who participate in the 2016 InterLab Study, were asked to follow the protocols below and to submit data using the forms and files provided. Teams could choose to follow one or both of these protocols. They were asked to measure fluorescence in five different E. coli constructs – each transformed with different plasmid (device).
Five plasmids were given to us from the iGEM. The first contained a high expression constitutive J23101 promoter, the second one contained a medium expression constitutive J23106 promoter and the third contained a very low expression constitutive promoter J23117. All three plasmids also contained GFP gene. Structure of the plasmids can be seen below.
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
Our team chose plate reader protocol and used 96-well plate for measurements. Following the given protocol, LUDOX –S30 had to be used as a single point reference to obtain a ratiometric conversion factor to transform our absorbance data into a standard OD600 measurement. LUDOX solution that we received was precipitated and we did not have enough time to have another sample of LUDOX sent. Therefore, we prepared LUDOX-S30 solution from Silica, fumed powder 0,007 μm. All the experiments were performed using this LUDOX solution.
FITC fluorescence standard curve was prepared by measuring FITC solutions of known concentrations. Standard GFP settings (Excitation: 485/20.0, Emission: 528/20.0) were used. We used 'BioTek Synergy H4 Hybrid Reader' for all fluorescence and absorbance measurements.
Escherichia coli TOP10 cells were transformed using these following plasmids:
Colonies from each plate were inoculated and incubated in the shaker at 37 ºC overnight. The following day, cultures were diluted to a target OD600 of 0.02 and grown. OD600 and fluorescence were measured using the same settings as were used for preparing standart FITC fluorescence curve. Samples of 1 % of total volume were taken after 0, 1, 2, 3, 4, 5, 6 hours of incubation at 37 ºC at 220 rpm. Samples were placed on ice and measurements were made at the end of sampling.
We had only one replicate of Device 1, where E. coli were growing successfully. Cell growth in other replicates was not detected. We indicated two measurement repeats on same replicate of Device 1. Otherwise standard deviation would be higher than Fl/Abs ratio.
We see a huge difference of measured parameters between positive and negative control.
All E. coli types (bacteria with different devices) were growing similarly, except for Device 1. Cells with Device 1 grew slower and final concentrations of E. coli with Device 1 was about 2 times lower.
Device 1 has the biggest fluorescence. Device 2 – lower, similar to positive control. Device 3 has the smallest fluorescence – similar to negative control. There is a direct relationship between absorbance and fluorescence. Fl/Abs ratio of different Devices decreases in order: Device 1, Device 2, Device 3. All the data of E. coli fluorescence are in the interval of standard FITC fluorescence curve.
GFP is expressed after transformation only. GFP expression is controlled by Devices 1, 2, 3 and Device with positive control. Fluorescence of GFP in genetically engineered E. coli rises with the increase of the cell number (absorbance 600). Some of the data indicates a decrease in cell quantity during incubation. This may be caused by measurement imperfection or cell settlement on the bottom of the well.
Fl/Abs ratio differs in samples of hours 0, 1, 2, while the same ratio of samples taken at 3, 4, 5, 6 hour stays more similar. This can be explained as standard error is smaller the longer the cells incubate.
Difference of fluorescence means different GFP concentrations in bacteria transformed with different devices. This can be explained by different promoter strength in each device. Data indicates that Device 1 had the strongest promoter, Device 2 had a weaker one and Device 3 had the weakest promoter. Results only prove the known hierarchy of promoter strength: J23101 > J23106 > J23117.