Reproducibility is the ability to recompute data, by following the exact same protocol and analysis methods of an experiment. The replicability or repeatability of a study is the chance that an independent experiment targeting the same scientific question will produce consistent results. Consistent findings from independent investigators and laboratories are the primary means by which scientific evidence accumulates for or against a hypothesis (Leek and Peng 2015). To maintain the integrity of scientific research and the public's trust in science, the scientific community must ensure reproducibility and replicability by engaging in wide-range interlaboratory studies. One such study is the International InterLab measurement study organized by Jacob Beal et al.
The InterLab study has been a part of the iGEM competition for four years, with 2017 being the fourth year, where teams participating in the competition can join in the effort to improve the measurement tools available to both the iGEM community and the synthetic biology community as a whole. The aim of this study is to develop a robust measurement procedure for the quantification of green fluorescent protein (GFP) expression and thereby provide researchers with a detailed protocol and data analysis form which yields absolute and interlaboratory - comparable units for GFP measurement.
This year, the fourth International InterLab Measurement Study aims to establish a standard GFP measurement protocol based on engineering principles that any lab with a plate reader can use. The procedure involves the measurement of GFP expressed by E.coli DH5α which has been transformed with plasmids expressing GFP, each of which has a different promoter or ribosome binding site. The measurement involves the use of a plate reader but, optionally, a flow cytometry measurement could also be performed for more data to support the standardizing procedure. Our team was more than excited to provide flow cytometry data but we couldn't get the appropriate calibration beads on time.
All plasmids used have the standard pSB1C3 backbone which has the high copy number pMB1 Origin of Replication resulting in 100-300 copies in each cell. The plasmids also have the Chloramphenicol acetyltransferase gene which detoxifies the antibiotic chloramphenicol providing resistance and therefore a selection ability of transformed bacteria when grown in LB-agar plates with it. The negative control only has an extra TetR promoter while the all other constructs have a promoter site, a ribosome binding site, GFP and two terminators built in them. The GFP gene is the same in all constructs (E0040) but the promoters and the ribosome bindings sites vary. The promoters were recovered from a library screened by Chris Anderson and each of them has a different strength. Three different ribosome binding sites were used; the first being a weak one based on Ron Weiss thesis (BBa_B0032), a strong one based on Elowitz repressilator (BBa_B0034) and the bicistronic design element Number 2 (BCD2) (BBa_J364100) designed by Mutalik et al (Mutalik et al. 2013).
The first step is to obtain a ratiometric conversion factor to transform absorbance data into a standard OD600 measurement through the use of LUDOX-HS40 (provided by iGEM) as a single point reference. This ratiometric conversion of 600nm absorbance into OD600 normalizes variances in data obtained from instruments with different sensitivities. The absorbance at 600nm is measured in both ddH2O and LUDOX and the corrected absorbance is calculated by subtracting the water reading. The Reference OD600 defined as that measured by the reference spectrophotometer (0.0425; value provided by iGEM) is then divided by the corrected absorbance and the result is the correction factor. By multiplying the absorbance at 600nm measured by each instrument, with the correction factor, all readings are now aligned to the reference spectrophotometer readings. Our plate reader was PerkinElmer EnSpire Multimode Plate Reader 2300 and measurements were made in the standard modes of the plate reader.
After the OD600 measurements are calibrated, we needed to create a standard curve for fluorescent measurements, again so that any differences in the instrument sensitivities, do not affect the results. By taking reads from a series of serial dilutions of a fluorescein sodium salt solution in the plate reader, the standard curve, with which GFP measurements can be normalized among different instruments, was created.
After these calibrations were made, the 8 devices had to be tested. All test devices were transformed into K12 DH5a E.coli by all iGEM teams participating, so that there are no variations in the expression systems between different strains. After plating onto agar plates with chloramphenicol as a resistance marker, two colonies were picked and grown overnight into LB Medium with chloramphenicol. Then they were diluted to an OD600 of 0.02 (conversion of Absorbance 600nm to OD was made by the correction from the LUDOX measurements) and allowed to grow for up to another 6 hours while taking time points every two hours. Samples were obtained every two hours and they were all measured together in the end by our plate reader. All of our results have been submitted to iGEM for further processing and comparison with data provided by the rest of teams.
We hope that our measurements will provide helpful data in this international study and confidently, along with the assistance of all other participating teams, create a standardized protocol for measuring absolute units of GFP expression for all researchers to use. We would like to encourage all teams in the upcoming years to participate in these studies, as together we can make large-scale experimental measurements seem like just another every-day experiment in the lab.
