BackgroundThis is the second time that we have participated in International Interlaboratory Study along with many other teams from around the world. Interlab studies is used to compare the results in different labs around the world. This study is organized by iGEM’s measurement committee in an effort to establish standardized, reliable and repeatable measurement tools for the iGEM community and the synthetic biology community as a whole. The objective of this year’s Interlab is to establish a GFP measurement protocol through measurement three calibration and cell measurement protocol.
OverviewAccording to the requirements, we completed calibration protocols first. We made three sets of unit calibration measurements: an OD600 reference point, a particle standard curve, and a fluorescein standard curve. In all three calibration measurements, we used the same plates and volumes in cell-based assays. For the plate reader our excitation and emission settings were 485 nm and 530 nm respectively (Same setting was used for all experiments below).In the process of the experiment, the measurement of the three calibration were remarkable smooth.But in restrict of experiment condition, we met a lot of difficulties in cell measurement experiment. In addition, cell measurement was not as good as expected at the first time, so we repeated the cell measurement experiment and sequencing was used to verify the success of the transformation. At last, we get the corrected results after repeat multiple times.
Results and discussionMeasurement of standard curveWe measured the LUDOX CL-X , Microsphere suspension and Fluorescein to obtain the following results. In this way we can transform absorbance data into a standard OD600 measurement and we obtain a Particle Standard Curve and a Fluorescence standard curve.Fig. 1 Using fluorescein to plot fluorescence standard curveFig. 2 Fluorescence is plotted in a log scale to demonstrate a linear relationship.Cell measurementDevices: Positive control: BBa_I20270、Negative control: BBa_R0040、Device 1: BBa_J364000、Device 2: BBa_J364001、Device 3: BBa_J364002、Device 4: BBa_J364003、Device 5: BBa_J364004、Device 6: BBa_J364005Microorganism: Escherichia coli DH5⍺ strainsThe protocol was easy to follow and the constructs were nicely expressed which makes our measurements more reliable and comparable. After 0 and 6 hours, we measured the OD600 and the fluorescence of the transformed cells according to the protocol. We obtained the following data from the measurement and use the three standard curves to calculate the data.
1 2 3 4 5 6 7 8 9 10 11 12 A Neg. Control 1-1 Pos. Control 1-1 Device 1 1-1 Device 2 1-1 Device 3 1-1 Device 4 1-1 Device 5 1-1 Device 6 1-1 LB+Chl Empty Empty Empty B Neg. Control 1-2 Pos. Control 1-2 Device 1 1-2 Device 2 1-2 Device 3 1-2 Device 4 1-2 Device 5 1-2 Device 6 1-2 LB+Chl Empty Empty Empty C Neg. Control 1-3 Pos. Control 1-3 Device 1 1-3 Device 2 1-3 Device 3 1-3 Device 4 1-3 Device 5 1-3 Device 6 1-3 LB+Chl Empty Empty Empty D Neg. Control 1-4 Pos. Control 1-4 Device 1 1-4 Device 2 1-4 Device 3 1-4 Device 4 1-4 Device 5 1-4 Device 6 1-4 LB+Chl Empty Empty Empty E Neg. Control 2-1 Pos. Control 2-1 Device 1 2-1 Device 2 2-1 Device 3 2-1 Device 4 2-1 Device 5 2-1 Device 6 2-1 LB+Chl Empty Empty Empty F Neg. Control 2-2 Pos. Control 2-2 Device 1 2-2 Device 2 2-2 Device 3 2-2 Device 4 2-2 Device 5 2-2 Device 6 2-2 LB+Chl Empty Empty Empty G Neg. Control 2-3 Pos. Control 2-3 Device 1 2-3 Device 2 2-3 Device 3 2-3 Device 4 2-3 Device 5 2-3 Device 6 2-3 LB+Chl Empty Empty Empty H Neg. Control 2-4 Pos. Control 2-4 Device 1 2-4 Device 2 2-4 Device 3 2-4 Device 4 2-4 Device 5 2-4 Device 6 2-4 LB+Chl Empty Empty EmptyFig. 3 Sketch map of plate patternFig. 4 The difference of fluorescence raw readings between colony 1 0h and 6hFig. 5 The difference of fluorescence raw readings between colony 2 0h and 6hFig. 6 The difference of fluorescence raw readings between 0h and 6hCompare the devices we can found that the different promoters will influence the expression of proteins. We can easily found that the promoters in test 1 and 4 is obviously stronger than the promoters in test device 3 and 6, for the GFP expression is higher than former.
ConclusionWe can find that the best fluorescence results is even higher than positive control. Device 4 shows the best fluorescence, but the absorbance values is not the highest. Device 3 shows us that the values of fluorescence almost no growth, but the absorbance values is the highest. At last, the data suggest that the Interlab study was successful and the protocol can be shared in the community and between the laboratories.
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