Team:HUST-China/InterLab Study

Team:HUST-China:Modeling



Interlab Study




SectionⅠ: Introduction

This year, iGEM invited and encouraged all teams to participate in the Second International InterLab Measurement Study in synthetic biology. We were required to measure the expression level of GFP when using three different promoters. Although It was the first time for our team to take part in Interlab Study, we successfully conducted the experiment and obtained the fluorescence data. Now, we are hoping that we can make contributions to Interlab Study.




Section Ⅱ: Provenance and Release

Individuals responsible for conducting InterLab study:

Create the devices: Zhi Zeng Conduct measurements: Guozhao Wu, Shuyan Tang and Zhi Zeng Process data: Shuyan Tang and Yee Zhan


Date of InterLab Study:

The measurement was obtained on August 27, 2015.


Did your team participate in the Extra Credit?

No. (Details can be found here: https://2015.igem.org/Tracks/Measurement/Interlab_study)


Do all persons involved consent to the inclusion of this data in publications derived from the iGEM interlab study?

Yes.




Section Ⅲ: Equipment Information

What type of incubator did you use to grow your cells?

THZ-D desktop constant temperature oscillator


If known, what was your incubator's throw (shaking diameter)?

Oscillation frequency: 20~350rpm

Amplitude: 26mm

Maximum capacity(each layer): 1000mlx6 or 500mlx9 or 250mlx12

Standard configuration: Spring rack

Dimension of the tray (mm): 400x340

Time range: 0~999minutes

Control temperature: (Environment temperature)Room temp+5℃~60℃

Temperature increment: 0.1℃

Inner temperature error: ±0.5℃

Display: LED

Input power: 350w

Size (mm): 650x500x480


What piece of equipment did you use to measure the devices?

FlexStation 3 Multi-Mode Microplate Reader


When was this equipment last calibrated?

August 1st, 2015


Who calibrated the equipment?

Tengteng Gong


What was the wavelength of light you used to excite the cells?

485nm


What was the filter/channel you used to capture the light emission from the cells?

Monochromators, tunable in 1.0 nm increments Wavelength range 400–750 nm


What was the sampling frequency?

50 times per second




Section Ⅳ: Protocol

Did you fill in the InterLab Protocol provided by the Measurement committee?

Yes (The InterLab Protocol is provided here: https://2015.igem.org/Tracks/Measurement/InterLab_Protocol)


Did you follow the InterLab Protocol provided by the Measurement committee?

Yes


How did you determine the final dataset that you are reporting?

Since theoretically the emission wavelength of GFP is up to 518nm and the test data showed 518nm got the peak signal, we chose Em518 data. We also tested LB medium as blank and the data was minus the blank.




Section Ⅴ: Measurement results for Interlab Study

Units Reported:

RFUs


We measured the three specific devices required by Interlab Study, using E.coli DH5α as our chassis.

1.Device 1: BBa_J23101 + BBa_I13504 (B0034-E0040-B0015) in the pSB1C3 backbone

2.Device 2: BBa_J23106 + BBa_I13504 (B0034-E0040-B0015) in the pSB1C3 backbone

3.Device 3: BBa_J23117 + BBa_I13504 (B0034-E0040-B0015) in the pSB1C3 backbone


We diluted each of our samples to an OD600 of 0.5 and we started measuring fluorescence when OD600 was within 5% of 0.5. We measured each sample in biological triplicates.


We obtained the fluorescence data by using 485nm as the excitation wavelength and 518nm as the emission wavelength. Measurement data were collected as relative fluorescence units.


Final fluorescence data were collected on August 27, 2015.

We divided fluorescence by relevant OD600 and used the results to draw the graph above. According to the graph, all the samples seemed to be normal except Device3: J23117+I13504. Sadly Device3 didn’t express GFP while Device1 had the highest fluorescence intensity, revealing that J23101 is a relative high strength promoter compared with J23106 and J23117.

The fluorescence results that we submitted to Interlab Study are below:

All the data below has been minus our blank control, LB medium.


The First Replicate The Second Replicate The Third Replicate
Positive Control: BBa_I20270 1346.963 1640.876 1265.491
Negative Control:BBa_K950001 -58.27 -63.088 2.847
Sample Biological Replicate 1 Biological Replicate 2 Biological Replicate 3 Mean Standard Deviation
Device 1: BBa_J23101+ BBa_I13504 4737.16 4864.736 4715.018 4772.305 120.498
Device 2: BBa_J23106+ BBa_I13504 1271.075 860.502 1581.901 1237.826 299.201
Device 3: BBa_J23117+ BBa_I13504 -31.803 -28.755 51.543 -3.005 50.803

We divided fluorescence by relevant OD600 and used the results to draw the graph above. According to the graph, all the samples seemed to be normal except Device3: J23117+I13504. Sadly Device3 didn’t express GFP while Device1 had the highest fluorescence intensity, revealing that J23101 is a relative high strength promoter compared with J23106 and J23117.





Section Ⅵ: Appendix

Raw data or our fluorescence measurement

Raw Data Collected
(Exciation Wavelength:485nm
Emission Wavelength:518nm)
Test Sample 1 Sample 1 Average Test Sample 2 Sample 2 Average Test Sample 3 Sample 3 Average Average Standard Deviation
LB Blank 956.328 835.504 924.036 905.289 902.121 930.765 888.7 907.195 844.124 885.305 841.321 856.917 889.800 40.598
DH5α 778.104 789.447 777.712 781.754 782.342 779.524 783.974 781.947 778.284 781.927 772.937 777.716 780.472 4.398
Positive Control: BBa_I20270 2550.953 2086.728 2119.077 2252.253 2805.108 2403.488 2435.617 2548.071 2326.168 2015.191 2025.865 2122.408 2307.577 254.267
Negative Control:BBa_K950001 886.838 800.389 853.831 847.019 849.775 807.333 875.214 844.107 911.623 792.095 875.574 859.764 850.297 39.571
Device 1: J23101+I13504 5833.139 5335.268 5758.94 5642.449 5769.227 5756.32 5790.246 5771.931 5569.237 5556.323 5590.245 5571.935 5662.105 151.731
Device 2: J23106+I13504 2253.464 2154.885 2120.743 2176.364 1776.392 1715.65 1811.049 1767.697 2448.247 2432.939 2435.268 2438.818 2127.626 279.005
Device 3: J23117+I13504 893.595 801.261 925.602 873.486 894.7 858.503 882.117 878.440 941.186 884.084 900.11 908.460 886.795 37.854