Team:SHPH-Shanghai/InterLab
1.Abstract
2.Measurement and Result
3.Discussion
Abstract
The goal of the fifth Interlab is to answer the following 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? By using two orthogonal approaches: Converting between absorbance of cells to absorbance of a known concentration of beads and Counting colony-forming units (CFUs) from the sample. The experiment was carried out in ECUST under their group members’ help. The strain we used for cell measurement was E. coli strain DH5-alpha and we transmitted 6 different plasmids, along with a negative control and a positive control in it.
The 8 plasmids are: BBa_R0040(Negative control), BBa_I20270(Positive control), BBa_J364000, BBa_J364001, BBa_J364002, BBa_J364007, BBa_J364008, and BBa_J364009 in the pSB1C3 backbone, all from the distribution kit.
Measurement and Result
Following the calibration protocol, we measured the absorbance of LUDOX CL-X as a reference. We also measured the 〖Abs〗_600 of dilution series of silica beads and fluorescence of dilution series of Fluorescein provided in the Interlab kit. The standard curves as well as OD600 reference points (Figure 1.1 1.2 1.3 1.4 1.5) reached by these measurements are used to help us check whether our dilution in the cell measurement is in linear range.
Then after the transformation of the 6 devices to the strain, we picked 2 colonies from each culture and incubated overnight. The next day, we measured their absorbance and fluorescence before and after a 6-hour incubation in the 96 well plate. (Figure 2.1 2.2)
The following figures are fluorescene per OD and per particle. (Figure 3.1 3.2 3.3 3.4)
The negative and positive control are adjusted to OD 0.1and then serial diluted until there are three samples for each colony with dilution factor 8×〖10〗^4, 8×〖10〗^5 and 8×〖10〗^6. The three samples are incubated on plate overnight. We did the colony count next day morning. The CFU can be calculated with the number of colonies using given equation: # colonies x Final Dilution Factor = CFU/mL (Figure 4)
Discussion
According to the strength of the promoter given on the website, the average fluorescence obtain from measurement should be device 4, 5, 1, 2, 6, 3 from highest to lowest. However, the data we obtained shows that the average fluorescence from high to low is device 1, 4, 5, 2, 3, 6. Between two devices the difference of the average value is rather small, so we assume that it might be within the range of uncertainties or some error occur when we were picking colonies to incubate them.
Another problem is that there is not a big difference between the fluorescence values measured in 0h and 6h. This may be because the fluorescence in 0h is measured after ice bath for 6 hours, and the ice bath may have failed to prevent the bacteria from growing, causing them to express fluorescent protein just like the 6h incubated bacteria. In addition, this may also account for the different order of highest level of fluorescence between 0h and 6h of the colonies, since bacteria are growing in ice bath and this growth may be unequal due to their position in ice. The solution in 0h will probably have a distinct fluorescence reading from the 6h solution if they were measured directly without ice bath.