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Introduction to the Lab Work
Objective
The goal of our wet lab work is to validate our software prediction. Since the terminator efficiency data available in literatures is very limited, we designed 100 terminators through the knowledge of terminator structure, and designed an experiment to measure terminator efficiency. By analyzing experiment results, we can adjust the parameters in our model to make it more accurate.
1, How to measure efficiency:
The figure above shows the plasmid designed to measure terminator efficiency. The terminator to be characterized would be flanked by gene sequences of two fluorescent proteins, GFP and RFP. By using flow cytometry, we could measure the fluorescence strength of GFP and RFP, so as to measure the terminator efficiency.
The terminator efficiency of a terminator is calculated with the formula:E = 1- S/T
where S is the fluorescence strength of GFP with terminator, and T is the fluorescence strength of GFP without terminator, which is the baseline strength. 2. Plasmid Construction3. Fluorescence strength quantification
We used flow cytometer to measured the fluorescence strength. Laser was used to be the luminous source and then radiated perpendicular to the sample flow. Under the irradiation of laser, cells expressed fluorescent protein. We calculated the average expression level of those cells and got the average strength of GFP. We also used fluorescence microscope to take a picture of bacterial culture.
4. Agreement with theoretical prediction
According to our experiment measured terminator efficiencies and our software predicted d scores, we created a fit curve to relate all the data. The corelation coefficient is 0.8. So we have achieved a good agreement between experimental efficiency and TTEC predicted efficiency.
5. Technical Standard
Our terminator efficiency measurement protocol has been submitted to Biobrick foundation as a technical standard(BBF RFC 90).
