Team:NTU SINGAPORE/InterLab
Interlab Measurement Study
Introduction
Our team is delighted to participate once again this year in the 4th InterLab Study. InterLab Study is one of the iGEM’s measurement committee’s effort to develop a reliable measurement procedure for iGEM and synthetic biology community, especially in fluorescence measurement. In the end, they seek to come up with protocols which will enable researchers to be able to compare their GFP measurement using plate readers more easily by converting into an absolute unit.
For this year, the committee focused aims to compare the measurement value when performed around the world with a standardized protocol. There are 6 devices which should be tested, along with a positive and a negative control. The test devices 1, 2, and 3 contains the same RBS element, while test devices 4, 5, and 6 contains another RBS devices, the bicistronic design element 2 (BCD2). Moreover, in three pairs of the test devices, which is test device 1 and 4, 2 and 5, 3 and 6, each is regulated by different promoters.
Methodology
OD600 reference point: We first established the correction factor to convert our absorbance at 600 nm (Abs600) into OD600 using LUDOX and H2O. The correction factor will later be used to determine OD600 of bacterial culture
Standardization of fluorescein curve: Then, a fluorescein standard curve was constructed to convert the GFP fluorescence readings in term of fluorescein concentration per unit reading (uM/a.u.). In order to make the curve, fluorescein salt from iGEM was diluted with DPBS into 1X concentration, equivalent to the concentration of 50 uM. Then, ten serial dilutions, each diluted with dilution factor 2, was performed in four replicates.
Cell culture measurements: The controls (positive and negative) and test devices (1, 2, 3, 4, 5, 6) were transformed into DH5a E. coli and two colonies were picked and grown in LB + Chloramphenicol overnight at 37 C and 220 rpm. Prior to inoculation, the plasmids had been sequenced to confirm that correct colony had been picked. Then, the colonies were diluted to OD600 of 0.02 in 12 mL LB + Chloramphenicol and subsequently sampled every 0, 2, 4 and 6 hours to be measured. To halt the cellular activity, the samples were put in ice before finally measured, The complete methodology follows from the iGEM protocol
Results
OD600 reference point
Using water (H2O) absorbance as a blank, the LUDOX absorbance was corrected to be 0.01075. The correction factor was obtained by dividing corrected absorbance with a reference OD value 0.0425 (specified by iGEM). Finally, the correction factor we got is 3.95.
The standard curve is shown if Figure 1. In average, the relation is 4.00 × 10−4 μM fluorescein/au.
Figure 1. The fluorescein standard curve.
OD600 measurement
Figure 2. OD600 readings for different devices, sampled up to 6 hours for every 2 hours.
The bacteria growth in the media as a function of time can be seen in Figure 2. All started from OD of approximately 0.01 and grow exponentially during the course of the experiment. However, bacterias harboring different test devices showed different growth kinetics; particularly, device 1 grew very slowly in both colonies, or even almost flat in colony 1. Then followed by the bacteria containing device 4, the positive control, and then the rest of the bacterias have almost similar growth kinetics.
Figure 3. Fluorescence intensity (FI) measurement for all test devices up to 6 hours.
With regards to fluorescence intensity, as seen in Figure 3, we can observe that the fluorescence intensity in device 1, 2, 4, 6, and positive control increases exponentially with time, while device 3, 6, and negative control have little fluorescence expression. However, it must be noted that this only measures the total fluorescence and can be influenced by the number of cells which were present. Hence, FI/OD600 is a better parameter to compare the expression of the test devices, as seen in Figure 4.
Figure 4. The fluorescence per unit OD600 in test devices. Error bars are ± s.d. (n = 4).
We can observe that from Figure 4, device 1 has the strongest fluorescence intensity per unit cell density compared with the other devices. Then, the FI/OD in device 1 is more than device 2 and device 3, consistent with the result in device 4, 5, and 6. Since each of the promoter in device 1 and 4 (J23101), 2 and 5 (J23106), 3 and 6 (J23117) are the same, we concluded that the order of the promoter strength is J23101 > J23106 > J23117. Moreover, we can see that the FI/OD in device 1, 2, and 3 are stronger than in device 4, 5 and 6, respectively. Hence, we can also draw that the presence of BCD2 reduces the fluorescence intensity.
Additionally, we can relate the FI/OD600 in Figure 4 with the growth kinetics of the bacteria in Figure 2. The FI ratio is very high, but the cell growth was also impaired. Furthermore, the cells with the lowest fluorescence, which is the negative control appear to have the fastest growth kinetics. Similar trends can be observed for the other devices as well. This suggests that expressing fluorescence may impose more stress to the cells, hence hasten its growth.
Conclusion
From the InterLab measurement, we concluded that the promoter strength order from the strongest is J23101 > J23106 > J23117. We also found out that the presence of BCD2 lowers the fluorescence intensity/unit absorbance in all the devices. Furthermore, expressing the fluorescence protein in excel could impose stress to the cells, hence showing slower kinetics.
