Team:SCAU-China/Interlab
The goal of the iGEM InterLab Study is reliable and repeatable measurement which is a key component to synthetic biology. We took part in the 5th International InterLab Measurement Study and the big question is
“Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD? ”
Parts
Interlab test devices used, as set by the iGEM measurement committee
| Device | Part Number | Location |
|---|---|---|
| Negative control | BBa_R0040 | Well 2D |
| Positive control | BBa_I20270 | Well 2B |
| Test Device 1 | BBa_J364000 | Well 2F |
| Test Device 2 | BBa_J364001 | Well 2H |
| Test Device 3 | BBa_J364002 | Well 2J |
| Test Device 4 | BBa_J364007 | Well 2L |
| Test Device 5 | BBa_J364008 | Well 2N |
| Test Device 6 | BBa_J364009 | Well 2P |
the preparation of competent cells(Escherichia coli strain DH5α) and their transformation according to the cell measurement protocol. Meanwile, we followed the Calibration Protocols supplied by the iGEM HQ to generate OD600 Reference point, Particle Standard Curve and Fluorescence standard curve to calibrate the plate reader(96 well plate) and then measured both the fluorescence and absorbance of our samples in 0-hour and 6-hour.
We used the set protocol that we were given so as to ensure everyone was doing the same thing. These can be found at this
Results and discussion
We submitted the iGEM’s completed spreadsheet to the measurement committee before the deadline, and received confirmation that the results were accepted on the 2018/7/30
OD600 calibration
| LUDOX CL-X H2O | ||
|---|---|---|
| Replicate 1 | 0.059 | 0.039 |
| Replicate 2 | 0.060 | 0.035 |
| Replicate 3 | 0.060 | 0.035 |
| Replicate 4 | 0.061 | 0.040 |
| Arith. Mean | 0.059 | 0.039 |
| Corrected Abs600 | 0.023 | |
| Reference OD600 | 0.063 | |
| OD600/Abs600 | 2.769 | |
Fluorescein standard curves
Particle standard curve
Our goal is to reduce lab-to-lab variability in fluorescence measurements, in order to test the variability of variantion in our sample, we caculate coefficient of variantion for both colony, and compare the CV value under different normalization method(Fluorescence per OD or Fluorescence per particle)
0th
6th
After 6h growth, we found that the variation of each type of cells(devices) is decreased, but the CV value of devices 4 and device5 are much more large than others. The variation of fluorescence per OD is much more smaller than fluorescence per particle. what’s more, we also found that colony2’s data is more steady compare with colony1 in most cases. for conveniently compare the normalized value with original value, we removed the data of devices4 and device5, and only compare the colony2 6-hour’s data.
According to the graph, we found that fluorescence per particle can decrease the variation but it depends on the devices. It seems that use OD value to normalize still better than particle. But one thing need to point out that too steady normalized value will loose sensitivity when test the influence of some factors. You can see this Excel File to check our raw and manipulated data.
School's name:SCAU
Member's name:SCAU
Designed by:SCAU