Team:NJAU China/Measurement
File Attached:
1.Sequencing File :
Device1.ab1
Device2.ab1
Device3.ab1
2.Protocol:
InterLab Protocol.pdf
Section I: Provenance and Release
Who did the actual work to acquire these measurements?Libo Sun--creating the devices, measuring them, and processing the data
Yang yang--creating the devices, measuring them
Zhiqin Chen--creating the devices, measuring them
Weiyi Wang--measuring them
Yuxing Fang--processing the data
On what dates were the protocols run and the measurements taken?
Cloning of constructs was confirmed by 10thAugust 2015. 24th August 2015 - all samples was measured usinga plate reader.
Do all persons involved consent to the inclusion of this data in publications derived from the iGEMinterlab study?
Yes.
Section II: Protocol
What protocol did you use to prepare samples for measurement?We used the protocol providedby the iGEM.
What sort of instrument did you use to acquire measurements?
TECHCOPM UV2300
TECAN infinite M200 with i-Control 1.9
How is it configured for your measurements?
This Fortessa has five lasers, of which we used two for our measurements. We used the 488 nm laser for our GFP measurements and the 561 nm laser for our RFP measurements (used in our controls). For the GFP measurements, we also used a FITC channel 530/30 filter while for the RFP measurements we used a PE-Texas Red channel 610/20 filter.
What protocol did you use to take measurements?
We used the protocol providedby the iGEM.
What method is used to determine whether to include or exclude each sample from the data set?
Plate reader: all samples were included in the data set.
What exactly were the controls that you used?
Positive Control: .E. coli K-12 DH5-alpha with BBa_I20270 transferred
Negative Controls: E. coli K-12 DH5-alpha without any plasmid added &E. coli K-12 DH5-alpha with BBa_R0040 transferred
What quantities were measured?
Spectrophotometer: Absorbance at 600 nm
Plate reader:Green fluorescence (485 ex/528 em)
How much time did it take to acquire each set of measurements?
The read and adjust for absorbance is about 60 s for the each test tube
The read for fluorescence is also under 90 s
What are the practical limits on the number or rate of measurements taken with this instrument and protocol?
Due to the short of supply of Corning® 96-well Clear Bottom Black Polystyrene Microplates of our supplier, we can only choose the WHB Black Polystyrene Microplates as an alternative. In this case, we can only measure OD600 useing a spectrophotometer.
Section III: Measured Quantities
Units
What are the units of the measurement?Equivalent ng/ml fluorescein per OD 600 unit
What is the equivalent unit expressed as a combination of the seven SI base units?
Absolute fluorescence could be expressed as equivalent moles of sodium fluorescein using the MW of 376.27.
Precision
What is the range of possible measured values for this quantity, using your instrument as configured for these measurements?For fluorescence on the plate reader, the sensitivity can be adjusted depending on the sample. Is the precision the same across the entire range? If not, how does it differ?
We prepare samples in triplicate in order to plot error bars in our result plots.
Accuracy
When was the instrument last calibrated?The plate reader was calibrated approximately 8 months ago.
Section IV: Measurements
We measured the following samples:• J23101 + I13504 (B0034-E0040-B0015)
• J23106 + I13504 (B0034-E0040-B0015)
• J23117 + I13504 (B0034-E0040-B0015)
*Build standard curve by measuring sodium fluorescein with certain concentrations(500, 375, 250, 125, 50, 25, 10, 5, 0 ng/ml). After subtracting average value of 0ng/ml. The data are shown as below. The function is y = 128.82x + 363.29.y is the indication of plate reader while the unit of x is equivalent ng/ml fluorescein.The curve is linear with R² = 0.9991.
| 500 | 375 | 250 | 125 | 50 | 15 | 10 | 5 |
| over | 48359 | 33172 | 16272 | 6442 | 3271 | 1280 | 671 |
*Convert all values to equivalent ng/ml fluorescein,including negative control group(with empty psb1c3)
*Calculate equivalent ng/ml fluorescein per OD by dividing OD 600 value of each sample.
*Eliminate background fluorescein by subtracting average value of negative control group
*Calculate S.D. value of each device to estimate the accuracy and precision of values.Calculate S.D. value of technical replicate to estimate the accuracy of machine.
|
1 |
2 |
3 |
Mean |
SD |
CV |
|
56.6178 |
55.1753 |
55.2194 |
55.6708 |
0.82038 |
|
|
36.5247 |
35.534 |
35.8372 |
35.9653 |
0.50762 |
|
|
48.9477 |
48.5902 |
49.6075 |
49.0485 |
0.5161 |
|
Mean |
47.3634 |
46.4332 |
46.888 |
46.8949 |
||
SD |
10.1398 |
9.99674 |
9.97319 |
8.70149 |
18.5553 |
|
1 |
2 |
3 |
Mean |
SD |
CV |
|
56.6178 |
55.1753 |
55.2194 |
55.6708 |
0.82038 |
|
|
36.5247 |
35.534 |
35.8372 |
35.9653 |
0.50762 |
|
|
48.9477 |
48.5902 |
49.6075 |
49.0485 |
0.5161 |
|
Mean |
47.3634 |
46.4332 |
46.888 |
46.8949 |
||
SD |
10.1398 |
9.99674 |
9.97319 |
8.70149 |
18.5553 |
6.61542 |
6.45525 |
7.28812 |
6.78627 |
0.44194 |
||
4.83218 |
4.06503 |
4.83218 |
4.57646 |
0.44291 |
||
9.17814 |
8.75727 |
9.45873 |
9.13138 |
0.35306 |
||
Mean |
6.87525 |
6.42585 |
7.19301 |
6.83137 |
||
SD |
2.1846 |
2.34626 |
2.31474 |
2.00507 |
29.3509 |
POS
34.5772 33.3178 33.9578
NEG
21.631 21.2461 23.1547