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