So far we have evaluated the function of all three components in our Box, with all of them exhibiting favaroble performances:
Now it is the very time to test the whole system!! :)
File:Blue light principle sjtu.gif As described in our [http://igem.bio-x.cn/Team:SJTU-BioX-Shanghai/Project/Light_sensor/Blue Project page], we have placed an gRNA that targets mRFP under control of blue light sensor.
After careful discussion, we decide to take up a three-step manner:
The Goal of the First Test is to discover a coarse range of adjustable region. We set up a gradient of electric current intensities, and during each time we provide a series of parameters into computer, thus giving different light intensities towards the test tubes. Inside test tubes are 5 mL cells with the above mentioned plasmid materials and after fixed times according to different needs. During the first time we want to figure out a good adjustable range of parameters, so we choose a wide range and hope to find the best region, which will be the foundation for the following accurate tests.
After a series of experiments and lots of data collection work we find that parameters under 40 show a good positive relationship with the mRFP amount and for those more than 50 a saturation phenomenon appears. After this test we immediately begin to design our second overall test hoping to get a meaningful result.
This time we choose parameters with the range of 0 to 35 and the interval is 5. The whole operation is the same with the first one except the input parameters. We expect there will be a very good positive relationship between the input parameters and the output amount of mRFP, and in fact we get the following result.
The result is very interesting and it is a little bit unexpected since there is not a strict linear relationship in this range. But we still get the wanted results that are the suitable region for our system. The best result appears between 15 and 35, and the linear relationship between input parameters and mRFP amounts is perfect for us to use.
In addition, we perform another control group to prove that it is not the light intensity that affect the final result. From the result of this control group we can see there is no obvious difference among different light intensities, which strongly proves it is our light-controlled CRISPRi system's correct function that leads to all these results.
Such linear relationship demonstrates a bright future of the application of our system, but this range is a little bit narrow. Holding the wish of widening the linear relation range, we test for the third time. Also it will be more persuasive to say that our system is reliable due to all the repeat tests give the right results.
Based on the previous tests and results we change the testing region to 15 to 45, and we hope to get the linear relationship. Data collecting work shows a good result again.
Up to now we have proved that the overall system can function well and we have already found out the range in which there will be a linear relationship between input parameters and the targeted gene amount, i.e. between the light intensities and mRFP amount. Based on our versatile and easy-to-use system, further test related to new application on genomic level about metabolic regulation is going to be performed.
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