Verified Components

So far we have evaluated the function of all three components in our Box, with all of them exhibiting favaroble performances:

• Verification of Luminous System
• Empirical Support of Light Sensors
• Verification of CRISPRi

Now it is the very time to test the whole system!! :)

Overall System

Blue 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:

First Test

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:

• when electrical intensities are under 40, these electrical intensities are in a nice positive relationship with the mRFP amount.
• And when electric intensities exceeds 50, the system becomes saturated.

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.

Second Test

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:

• The correlation is indeed positive between 15 and 35. And to our delight, the relationship is approximately LINEAR.
• But the correlation is, on the contrary, negative under 15.

Together with the case study above, we have set up strict BLANK controls: these control strains do not contain gRNAs. We paralleled the control group with the case group.
We proved that, mRFP amount in the control group would not vary significantly with the increase of light intensity (that is, with the increase of our UI input), which is a strong evidence that those relationships we observe in case groups is due to the correct function of our overall system, not just a effect of varied lights.

Despite the pity that the favorable range is not as wide as we thought, such linear relationship between 15 and 35 demonstrates a bright future of our system. Wishing to exactly determine the adjustable region (and perhaps, linear region), we conducted a third test. Furthermore, it will be more persuasive to provide a more accurate range in which our Box can be correctly utilized.

Third Test

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.

Red System

We also successfully constructed a red system, which can regulate the expression of genes with different strength of red light. To verify the validity of our red system, we use luciferase as a reporter gene. The protein luciferase can emit light with the existence of luciferin.

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. We has repeated it for 4 times, and the results are the same. The results are chiefly as follows.