Team:Rice/InterLab
INTERLAB
Introduction
This year, teams participating in the Interlab Study had to transform eight DNA test devices (see Table 1 below) provided in the distribution kit into E. coli DH5α cells and measure GFP fluorescence of the cells at four different time points. The purpose of the Interlab Study was to assess the reproducibility of fluorescence measurements across different labs and identify possible sources of variation in results obtained when following a common protocol. To determine possible sources of variation, the six test devices include all of the combinations of 3 related constitutive promoters with either the RBS B0034 or the bicistronic design element J364100.
Results
Standard Curves Generation
The Interlab Study protocol required two standard curves to be generated: Ludox OD600 reference point and fluorescein fluorescence standard curve. To generate the OD600 curve, we added each LUDOX and water to 4 wells of the 96 well plate and measured OD600 with a plate reader. The results are shown in table 2.
To produce a fluorescein fluorescence standard curve, we made 4 sets of fluorescein serial dilutions in a 96 well plate by preparing 1x fluorescein solution in PBS and adding the appropriate amounts of fluorescein and PBS into 48 plate wells (see our notebook for more details). The curve generated is shown in figure 1 and figure 2.
Figure 1 : Fluorescein Standard Curve
Figure 2 : Fluorescein Standard Curve (log scale)
Fluorescence Measurement
On the first day of the study, we transformed 8 Interlab parts provided in the distribution kit, following the steps of the transformation protocol. After the overnight culture of the plated transformed cells, we observed that the transformations were successful for the test devices 1, 2, and 5, but no colonies were present on the plates corresponding to negative control, positive control, and test devices 3, 4, and 6. We repeated the transformations for those samples and colonies were observed for all of them after another overnight culture. The next step of the Interlab Study was to prepare liquid cultures from colonies formed on the LB agar plates. We inoculated two colonies from each plate in LB with chloramphenicol (17 μg/mL) for the total of 16 liquid culture samples. The cultures were incubated overnight in 37 °C shaking incubator. In the morning, we retrieved the cultures from the incubator and measured OD600 of the cultures using a spectrophotometer. The results of the measurements are shown in Table 3.
Using the calculation sheet provided in the instructions, we diluted the samples to the OD600 of 0.02 with LB containing chloramphenicol (17 μg/mL). We then filled columns 1 through 8 of a 96 well plate with 100 μL of liquid culture per well (4 replicates for each sample). Column 9 was filled with chloramphenicol solution in LB. The flasks with diluted cultures were covered with foil and placed into 37 °C shaking incubator. Fluorescence and OD600 of the samples were measured with a plate reader at the following settings: 600 nm absorbance wavelength, 395 nm excitation wavelength, 509 nm emission wavelength. The same procedure as described above was repeated after 2, 4, and 6 hours of incubation from the first measurement. Identical plate reader settings were used for all measurements. We then exported the data into the provided Excel sheet to obtain values adjusted using the standard curves. Figure 3 provides a graphical representation of our fluorescence data for each of the test devices and controls; the values presented are an average of the fluorescence data for two colonies from each transformed construct.
Figure 3 : Average Fluorescence vs. Time
Discussion
The significance of our results cannot be determined because the bacteria transformed with the Negative Control plasmid (which does not contain a fluorescent reporter protein) produced a fluorescent signal over time.This unexpected result calls into question the validity of all of our other samples. Continuation of this study would involve repeating all transformations and fluorescence/absorbance measurements.
