The past interlaboratory studies have established a baseline for replicability of fluorescence measurements and identified likely key
sources of error, which have now been published [1], under review, or in preparation.
The goal for the fifth InterLab is to correct systematic variability, in order to achieve the InterLab protocol’s full potential as
an engineering discipline. Our team members are excited to participate in the InterLab Study, trying our utmost to provide data
helping answer the question: can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count
or colony-forming units (CFUs) instead of OD?
During the experiment, we carefully followed the Plate Reader and CFU protocol. After days of hard work, our data and forms are
completed and have been accepted by the 2018 Measurement Committee.
Team:SYSU-CHINA/InterLab
Interlab
Our work and results for Interlab measurement
The measurement was done by carefully following the Plate Reader Protocol from
https://2018.igem.org/Measurement/InterLab/Plate_Reader.
InterLab Measurement Kit
Eight devices to be tested:
| Negative Control (BBa_R0040) |
| Positive Control (BBa_I20270) |
| Test Device 1 (BBa_J364000) |
| Test Device 2 (BBa_J364001) |
| Test Device 3 (BBa_J364002) |
| Test Device 4 (BBa_J364007) |
| Test Device 5 (BBa_J364008) |
| Test Device 6 (BBa_J364009) |
Bacteria strain: Escherichia coli DH5-α
LB medium with Chloramphenicol(25ug/mL)
Plate reader: BioTek? Synergy H1
Black 96 well plate with flat, transparent bottom
OD600 reference point was obtained by measuring the Abs600 of H2O and LUDOX CL-X from InterLab Measurement Kit, followed by data processing.
Particle standard curve was obtained by serial dilution of the monodisperse silica microspheres followed by measurements using plate reader.
Fluorescein standard curve was obtained by serial dilution of the fluorescein solution followed by measurements using plate reader.
Plasmids of each device was re-suspended from Distribution Kit and used to transform Escherichia coli DH5-α. 2 colonies from each plate was picked and inoculated in 5ml LB medium with Chloramphenicol. The overnight cultures were each diluted to a target OD600 of 0.02 in 12ml LB medium. The diluted cultures were incubated at 37℃ and sampled at 0 and 6 hours post incubation. The OD600 and fluorescence intensity was measured at each time point.
Following the dilution way in protocol, we got perfect particle standard curve and fluorescence standard curve. These two standard curves actually provide solid foundation for our further experiment.
Figure 1. Particle Standard Curve with all data points using linear regression method.
y = 4^10-9x + 0.0438，R2 = 0.99793
Figure 2. Fluorescence Standard Curve with all data points using linear regression method.
y = 1986.9x + 112.09，R2 = 0.99842
The following graph shows the OD600 of each device at two time points. All the samples grow with a similar trend. There’s no significant difference among each sample’s OD600 value increase.
Figure 3. Histogram of Different Cultures’ OD600 at Each Time Point
The following graph shows the fluorescence intensity of each device at each time point. The fluorescence of Positive Control, Test Device 2 and Test Device 4 grow stronger obviously after 6 hours, while others’ fluorescence levels only have minor changes or maintain the original level. These differences may be caused by diverse promoter or RBS in each device.
Figure 4. Histogram of Different Cultures’ Fluorescence at Each Time Point.
During the experiment, we observed that the OD600 values and fluorescent levels of samples could change significantly after standing
for 20min or longer time. We speculate that the deviation of test results may be caused by bacterial cell growth or deposition or
some other unknown reasons. Therefore, a timely sample measurement is pretty pivotal to the success of the experiment.
The time delay always happens when the testing instrument has not been adjusted before or the instrument is being occupied by other
students. According to our experience, we hold that it is necessary to emphasize the importance of the measurements timeliness in the
next-year InterLab protocol.
Beal, J. et al. Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli. PloS one 11, e0150182, doi:10.1371/journal.pone.0150182 (2016).
