Team:HZAU-China/Interlab
Interlab
Abstract
iGEM teams from all around the world were invited to participate in InterLab Measurement Study, the goal of which is to obtain fluorescence intensity data of three specific devices. This year, we take part in InterLab Measurement study again. The devices have been assembled by the iGEM authority to express GFP protein through three different promoters. The study include three parts: measuring OD600 reference point with LUDOX-S30, making a FITC fluorescence standard curve and measuring OD600 and fluorescence of cells that contain the assembled plasmids. E.coli DH5alpha strain are choosen as the chassis. And we opt for plate reader and 96 well plate to take cell measurement. The devices are listed below:
Device 1: J23101.B0034.E0040.B0015 in pSB1C3
Device 2: J23106.B0034.E0040.B0015 in pSB1C3
Device 3: J23117.B0034.E0040.B0015 in pSB1C3
Positive Control Device: I20270 in pSB1C3
Negative Control Device: R0040 in pSB1C3
Materials and methods
OD600 Reference point
We used LUDOX-S30 as a single point reference to obtain a ratiometric conversion factor to transform our absorbance data into a standard OD600 measurement. It has been finished with the materia of LUDOX-S30 and water.
FITC fluorescence standard curve
In this part, FITC was resuspended in 1ml 1×PBS and incubate the solution at 42 ℃ for 4 hours and then be diluted to half concentration with another 1 ml 1×PBS. To obtain the fluorescence for FITC concentration as a standard curve, we perform a serial dilution from 100% to 0% in 12 wells. Finally, we obtained the FITC fluorescence standard curve. You can find it below:
Cell measurement
In the cell measurement experiment, we did not totally follow the protocol that was suggested by iGEM official since we did not set biological replicates but set technical replicates. We could not get positive data if we put the samples in the ice and measure it after several hours. In the alternative protocol, 0.5ml eight-hour cultures are added to 100ml flashes , each of which contains 50ml LB medium +Chloramphenicol. Then we incubated the cultures at 37℃and 220 rpm. After that, OD600 of the cultures are measured by spectrophotometer every 30 minutes until the OD600 reached 0.4. We diluted the cultures to a target OD600 of 0.02 with 20 ml LB medium in 50 ml falcon tube and then poured half of the medium to a vacant falcon tube.
When taking samples, we directly placed the 96-well-plate in the ice and added 100ul cultures to the wells at the corresponding moment of 0,1,2,3,4,5 and 6 hours of incubation . We measured the samples with plate reader right after we added them to the wells. Keeping incubating the cultures at 37℃ and 220 rpm and measuring the samples every one hour. Finally, we measured them by plate reader for 7 times and get our data of OD600 and fluorescence of the samples. Otherwise, we calculate the expression quantity by divide OD600 with data of fluorescence.
A=correction factor ×（A1－A0）
F=F1-F0
A1 and A0 are the raw data of Abs600 of the incubation and LB respectively. F1 is the data of fluorescence of incubation and F0 is of LB. We need the final result of F/A to analyze and make a graph.
Here is the protocol of Multi-Mode Reader：
Attention:The settings of the plate reader are the same among these three parts of measurement.
If you want to see the more details of the experiment, please download this pdf. provided by the iGEM official. However, we did a little different from that in the cell measurement part.
Results
We obtained the correction factor by this equation below:
Reference factor(provided by the iGEM official)/(Abs600 of LUDOX-S30－Abs600 of water ). It is 2.269231 which can convert Abs600 to OD600.Then we made line charts and a histogram by analyzing the data read by the plate reader.
Figure1. The graph above shows the variation of OD600 of the incubation from 0h to 6h.
Figure2. This graph is about fluorescence changing during the six hours.
Figure3. In this histogram, we can see the result of FI/Abs600 which indicates the expression of fluorescence of each cell.
Discussion
We are confused about the reason why we can not get the positive data by using the protocol provided by iGEM official. But the revised protocol really worked. We speculate that it may due to the degradation of the fluorescence since the samples collected at the first several hours are put in the ice for a long time. In the final result, we find the intensity of the three promoters in our measurement does not fit the data provided by the iGEM official. Compared to them, J23106 is the strongest one among these three during the first hour to the sixth hour. However, according to the iGEM official data, the intensity data of fluorescence of the E.coli with Device1 is higher than with Device2 and Device3.
Reference:
Jacob Beal, Traci Haddock-Angelli, Markus Gershater, Kim de Mora, Meagan Lizarazo, Jim Hollenhorst, Randy Rettberg, iGEM Interlab Study Contributors.(2016).Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. Coli. PLoS One, 11(3), e0150182.
