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!! :)
As described in our [http://igem.bio-x.cn/Team:SJTU-BioX-Shanghai/Project/Light_sensor/Blue Project page], we have placed a gRNA that targets mRFP under control of blue light sensor.
After careful discussion, we decide to take up a three-step manner:
GOAL: To discover a coarse range of adjustable region.
We set up a relatively sparse gradient of electric intensities on the User Interface. And corresponding blue lights would be produced into culture tubes, inside which there are 5mL culture of E. coli cells bearing above mentioned plasmids.
We check the result at regular intervals.
After a series of careful experiments and data collections, we successfully confirmed that:
As soon as we have acquired this suitable range of UI inputs, we immediately continue to our Second Test, in which we would like to further verify this range, deriving a detailed relation curve in this region.
GOAL: To further verify this adjustable range, deriving a more accurate relation curve in this region. According to results from our first test, we set up a finer gradients: from 0 to 35, with an interval of 5. All other conditions are the same with the First Test. We expect a good positive correlation between the amount of mRFP, the output, and the our UI inputs. But surprisingly, the result is a little bit different from our prediction:
The correlation between the output is not always positive in this range:
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.
To verify the validity of our red system, we use luciferase as a reporter gene. We observed obvious difference of the strength of fluorecence between the sample with and without the radiation of red light, which means that we can get larger amount of products under stronger red light. The result is chiefly as follows
"
