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Revision as of 16:12, 28 October 2017
「Interlab」
Introduction
It is significant for synthetic biology to develop a reliable and repeatable measurement, the same to all the other engineering disciplines. We HUST-China have volunteered to test some RBS devices (BCDs) that are intended to make gene expression more precise and reliable by measure the expression level of GFP, in order to help the iGEM community collect data about how reliable will these devices turn out to be in labs around the world.
Provenance and Release
①Individuals responsible for conducting InterLab study
Individuals | Interlab Part |
---|---|
Kangyuan Yu, Haibo Huang, Ziyang Xiao, Shaofeng Liao | created the devices |
Efan Wang, Long Cheng, HuiPing Shi | conducted the measurements |
Efan Wang | processed the data |
②Corresponding email
Individuals | Emails |
---|---|
Efan Wang | erfan@hust.edu.cn |
Kangyuan Yu | 985930862@qq.com |
Haibo Huang | u201512127@hust.edu.cn |
Ziyang Xiao | 372657289@qq.com |
Shaofeng Liao | 15827233830@qq.com |
HuiPing Shi | 172295915@qq.com |
Long Cheng | u201512127@hust.edu.cn |
Chassis and Safety
What chassis did you use?
Escherichia coli DH5alpha
What Biosafety Level is your chassis?
BSL1
What PPE did you utilize during your experiments?
Tianming gloves
Songxinjiujiu labcoats
Instrument
What instrument did you use during your measurements?
plate reader
Please provide the brand and model of your instrument.
Flexstation 3
Calibration Protocol
A1. Protocol for Optical Density (OD600) Standard Measurement
Did you use pathlength correction during measurement?
Yes
Number of flashes per well
6
Orbital averaging (mm)
600
What temperature setting did you use during the measurement?
22℃
What type of 96-well plate did you use?
Black plate (preferred)
Did your plate have flat-bottomed or round-bottomed wells?
Flat
A2. Measurement Steps
B1. Protocol for FIuorescein Fluoresence standard curve
Did you use pathlength correction during measurement?
Yes
Number of flashes per well
6
What gain setting did you use?
Automatic
If you used a filter, what light wavelengths did it pass?
530nm
Emission wavelength
530nm
Excitation wavelength
485nm
Fluorescence reading
Bottom optic
What type of 96-well plate did you use?
Black plate (preferred)
Did your plate have flat-bottomed or round-bottomed wells?
Flat
What temperature setting did you use during the measurement?
22℃
B2. Measurement Steps
Part 1: Prepare the Fluorescein stock solution
Measurement work flow:
Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.
![demo1 demo1](https://static.igem.org/mediawiki/2017/f/f8/2017_HUST_China_interlab_img_001.png)
Part 2: Prepare the serial dilutions of Fluorescein
Cell Culture Setup and Measurement
Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.
![demo1 demo1](https://static.igem.org/mediawiki/2017/e/e6/2017_HUST_China_interlab_img_002.png)
Transformation:
- control
- Negative control
- Test Device 1: J23101+I13504
- Test Device 2: J23106+I13504
- Test Device 3: J23117+I13504
- Test Device 4: J23101.BCD2.E0040.B0015
- Test Device 5: J23106.BCD2.E0040.B0015
- Test Device 6: J23117.BCD2.E0040.B0015
Cell growth:
Cell growth, sampling, and assay
The initial OD600 measurement of our overnight cultures.
![demo1 demo1](https://static.igem.org/mediawiki/2017/1/1a/2017_HUST_China_interlab_img_table1.png)
What type of media did you use for this step?
Luria Bertani
What type of vessel or container did you use to grow your cells?
50 ml Falcon tube
What temperature setting did you use during the measurement?
22℃
What type of 96-well plates did you use?
Black plates with transparent/clear bottom (preferred)
Flat
Measurement
- Measure OD and fluorescence of all samples
- Import data into blue cells in Excel (cell measurement) sheets provided
Suggested Plate Layout for 96-well Plate
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![demo1 demo1](https://static.igem.org/mediawiki/2017/8/87/2017_HUST_China_interlab_img_003.png)
Interlab Resultn
OD600 Reference Point
![demo1 demo1](https://static.igem.org/mediawiki/2017/5/52/2017_HUST_China_interlab_img_table2.png)
Fluorescence standard curve
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![demo1 demo1](https://static.igem.org/mediawiki/2017/a/a1/2017_HUST_China_interlab_img_table3.png)
NOTE: 50uM Sample exceeds the range of measurements
OD600 Reference Point
![demo1 demo1](https://static.igem.org/mediawiki/2017/d/d1/2017_HUST_China_interlab_img_table4.png)
![demo1 demo1](https://static.igem.org/mediawiki/2017/d/df/2017_HUST_China_interlab_img_table5.png)
Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.
![demo1 demo1](https://static.igem.org/mediawiki/2017/b/b3/2017_HUST_China_interlab_img_table6.png)
Final scaling level determined from medium-high points likely to be less impacted by saturation or pipetting error.
If needed, you can shift which points are used, but it is likely better to correct instrument settings and protocol.
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![demo1 demo1](https://static.igem.org/mediawiki/2017/b/b8/2017_HUST_China_interlab_img_table7.png)
Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.
![demo1 demo1](https://static.igem.org/mediawiki/2017/e/ea/2017_HUST_China_interlab_img_table8.png)
Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.
![demo1 demo1](https://static.igem.org/mediawiki/2017/d/d2/2017_HUST_China_interlab_img_table9.png)
Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.
![demo1 demo1](https://static.igem.org/mediawiki/2017/2/2b/2017_HUST_China_interlab_img_table10.png)