Team:SUSTech Shenzhen/Demonstrate

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Demonstration

With the data obtained in the RESULT part, we can demonstrate that the C-hoop system is accurately characterized and worked as expected in complex and more realistic condition. The multi-scale experiments results confirm that our system is functional and basically achieved all aspects of our original objectives.

Successfully Confirmed the Function Stability in Mammalian cell line

The “LightOn” system is successfully constructed in HeLa cell line and stably performed the desired function LightOn system is a light-switchable transgene system derived from prokaryotic species. Thus, to design a vector for it to stably work in mammalian cell is necessary. After plasmid construction and lentivirus transfection, FACS data shows that the LightOn system is successfully constructed into HeLa cell.

plasmid construction
Figure 1. Plasmid construction
Lentivirus transfection
Figure 2. Lentivirus transfection
The results of infection
Figure 3. The results of infection

Accurate Chracteriazation of LightOn system in various scales

We successfully characterized the dynamic property and found the optimal illumination condition for building further control model. The result data demonstrated the multi-level cell behavior changed respectively after the light induced transcription factor is activated.

Figure 4. qPCR test on mRNA of hGluc transcription in 60h light induce experiment
Figure 5. qPCR test on mRNA of hGluc transcription in light-dark conversion system
Figure 6. Flow cytometry test on mRuby translation
Figure 7. Chemiluminescence test on hGluc secretion

From these multi-sacle data, we demonstrated that:
(1).The optimal intensities(102.4uw) and illumination time(48h)
(2). For the qPCR test, RNA has shorter half-life, thus its change is more dynamic compared to secreted protein. Hence, it provide some characteristics of RNA dynamics for further modeling.
(3). For the flow cytometry test, we obtained characterization data of translation process.
(4). For the hGluc chemiluminescence test, we characterized the secretion process after protein translation. This set of data enable us to characterize he relationship between light exposure and Gene expression on multi-level (transcription level, translation level and secretion level), which is vital for further acquisition of experimental parameters and model constructions.


Demonstration of our control system's function in a more realistic and complex condition

3D-culture of HeLa cell

We cultured a 3D Hela cell mass to reflect the in vivo condition and we conformed functional stability in this simulative physiological condition.

Fig:3D cell mass in minipit
Figure 8. 3D cell mass in minipit
Fig:comparison of intracellular signal with extracellular signal, both the two kinds of the sinals reach their summit at the light power of 89.6μW
Figure 9. Comparison of intracellular signal with extracellular signal, both the two kinds of the sinals reach their summit at the light power of 89.6μW

By testing 3D cultured cell mass, we further confirmed the stability of our system in a more complex condition.

Cell immigration test

 We tested HL60 cell immigration toward our light-induced HeLa interkeukin-8(IL-8) secretion in a simulative immune reaction.

Figure 10. Chemotaxis assay sketch map
Figure 11. Chemotaxis assay plate

 We set up three groups of experiments on the Transwell Assay plate, which are positive control group, experimental group and negative control group, respectively, adding IL-8 on both sides, adding IL-8 on one side and adding culture medium on both sides. Then, we used the living cell imaging equipment to continuously track and photograph the cells in three environments for 15 hours.

 After analyzing the photos taken for a long time, we found that the cells in the experimental group had an obvious migration tendency to the IL-8 side; in the positive control group, we found that the cells had a relatively obvious movement tendency to both sides; in the negative control group, we found that the movement of the cells did not have regularity, and did not tend to one side of the significant movement.

 Therefore, we have proved that the IL-8 secreted by our cells has the expected biological activity through cell migration experiment. IL-8 plays an important role in cell recruitment in the immune environment, which proves that we have achieved the expected effect on the expression and secretion of target genes in cells by artificial regulation.

Verification of the Predictive and Control Model

We tested the accuracy of our predictive and control model with a 150-hour-long regulation test. Although Our model-based system is currently not perfect which may due to the step of control sequences, we will further modify our control functions to achieve ideal effects. During the regulation test, relevant control sequences that are returned from our simulation algorithm are input into the computer to make the concentration of hGluc fluctuate within a certain range, which is a proof-of-concept that we can effectively control its concentration changes under in vitro conditions. And the results show that the rise time (the time required to reach the first upper bound) is around 18 hours while the time required to reach the first lower bound is around 70 hours.

Figure 4. Regulation test
Figure 12. Regulation test