Team:SUSTech Shenzhen/Results
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Results
Result on construction of LightON system in HeLa cells
To characterize the gene expression in mammalian cells, we first constructed two plasmids, 5xUAS-mRuby-P2A-hGluc and EF1α-GAVPO-Bla. We chose hGluc because it has a low molecular weight and it is easy to measure the concentration. Hence, we can use it to mimic the production and secretion process of low molecular weight proteins such as cytokines. Also, we construct a plasmid 5xUAS-mRuby-P2A-hGluc-P2A-IL-10 to test the accuracy of regulation in a more realistic condition. Next, we constructed a plasmid 5xUAS-mRuby-P2A-hGluc-P2A-IL-8. Since IL8 can promote the activation and chemotaxis of neutrophil, it can be detected by observing the migration of neutrophil which is a common process in inflammation and immune reaction.
Next we Use lentivirus to transfect them into hela cells which is more easily cultured in vitro.And we also use Blasticidin to pick transfected cells and further identify monoclonal cells by using FACS.
We also test whether Hela cells are successfully transfected after being Illuminated for 24h through observing red fluorescence in Flow Cytometer. FACS data shows that the LightOn system is successfully constructed into HeLa cell.
Result on characterization of light-induced gene expression in Hela cells
In order to actualize precise regulation of light-induced cell behavior, we characterized the LightOn system’s function on inducing downstream expression process at different levels.
1.Determination of optimal illumination conditions
In the first place, we need to know the optimal condition of blue light exposure, then we can precisely decide the range of light condition in latter experiments. We designed a series gradients of illumination time and light intensity to set different illumination conditions. Then we obtained data of target protein (interleukin-10) secretion level and cell metabolic condition (using cck8 method) under different conditions.
(i): Result of Relationship between IL-10 expression and light intensity:
We measured the red fluorescence level which shows high positive rate after illuminated for 24h. Also through applying continuous illumination to HeLa cells, we have obtained the optimal intensities(102.4uw) and illumination time(48h).
2. Characterization of the whole expression process at different levels (transcription, translation, and secretion)
After obtaining the optimal condition of illumination, we are able to efficiently quantitatively characterize the whole expression process at different levels. We characterized the transcription process by testing the change of RNA through quantitative PCR. Next, we characterized the translation process by testing the dynamic change of mRuby through flow cytometer. The final step is to characterize the secretion process. Since we have chosen hGluc as our target product, we did it by measuring the chemiluminescence value.
(i): Result of qPCR test on transcription characterization
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1). 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. 2). For the flow cytometry test, we obtained characterization data of translation process. 3). 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.
Result of Functionality Test in 3D-culture cells
In the repeated light intensity and time gradient experiments as well as detection experiments, the cells we used are all in 2D culture state - that is, the cells grow in a monolayer state in a culture dish. Considering that the future application scenarios of the project may be more applied to animal model and theraputic studied, it is very important whether the blue light used in the experiment can pass through multiple laters of cells and effectively stimulate GAVPO transcription factor for transcription.
Based on the above point of view, we used 3D printing mold to build "mini-pit" on hydrogel. By adding the cells to the pressed hydrogel, we can get the ideal cell mass in the pit.
After the gradient experiments for exploring the best conditions, we found that the ideal cell mass can be achieved by the following methods:
1.1.2% hydrogel
2.550μL hydrogel per well in the24-well plate
3. Penicillin and streptomycin were incubated for more than 2 hours.
4.Add 1.5*10^4 cells per well in the 24-well plate.
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In order to verify the ability of cell 3D mass to produce and secrete target genes (cytokines) under the stimulation of GAVPO promoter, we tested the function of cell 3D mass at about 102 μ w of light power. We set up 6 groups of parallel control experiments, and the cells were cultured in 6-well plates. In the subsequent analysis of the results of flow cytometry, we found that the intensity of the target gene expressed by the cell mass in 6 groups was almost the same, and compared with the cells in 2D culture state, the expression intensity of the target gene in the same light condition was almost the same. It shows that the function and stability of cell 3D mass have been verified.
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In order to verify the effect of light intensity on the expression of target genes in cell 3D mass, we set up a light intensity gradient experiment of 0 μ w-150 μ W. In this experiment, we detected intracellular signal and extracellular signal respectively. In the intracellular signal part, we detected the fluorescence intensity of mRuby in the cell. We found that there were two kinds of cells with different fluorescence intensity in the flow cytometry map. Based on this, we speculated that the cells located in the outer layer were easier to obtain light, the target gene stimulated by GAVPO promoter was more transcripted and translated, the cells located in the inner layer received less light, and the expression level was lower. In addition, in the experiment of 0 μ w-150 μ W, the intracellular mRuby signal intensity firstly increased and then decreased, reaching the peak at 89.6 μ W. In the extracellular signal part, we detected hGluc secreted by cells, and the overall trend of signal intensity was still rising first, then falling, the peak value was at 89.6 μ W.
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On the whole, cell 3D culture test not only verifies the function and stability of cells under 3D mass, but also embodies the unity of extracellular signal and intracellular signal in a same trend. Both the intracellular signal of mRuby and the extracellular signal of hGluc reach their summit at the light power of 89.6μW.
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Determination of cell activity
In the process of cell experiment, we realize that the metabolic activity state of cell itself has an important influence on the production of target gene products (cytokine / luciferase). That is to say, when the cell is in malnutrition or dying state, its ability or tendency to transcribe or translate target gene will be greatly weakened, because this nonessential gene can not meet the most basic survival of cells' demand. Therefore, it is very important to understand the production and secretion of cytokines under the condition of human regulation, and the influence of cell metabolic activity stste and cell metabolic activity state on the expression of target genes.
In order to describe the metabolic activity state of cells, we selected Cell Counting Kit-8 to test cells. Cell Counting Kit-8 (CCK-8) allows sensitive colorimetric assays for the determination of cell viability in cell proliferation and cytotoxicity assays. Dojindo’s highly water-soluble tetrazolium salt, WST-8, is reduced by dehydrogenase activities in cells to give a yellow-color formazan dye, which is soluble in the tissue culture media. The amount of the formazan dye, generated by the activities of dehydrogenases in cells, is directly proportional to the number of living cells. The detection sensitivity of CCK-8 is higher than the other tetrazolium salts such as MTT, XTT, MTS or WST-1.[1]
Through the data integration and analysis in the later period, we took the beginning of the illumination experiment as the benchmark. With the growth of the illumination time, the cell metabolic activity state continued to decline. The cell state decreased significantly in 0-20h. After 20h, the cell metabolic activity state was only 50% of the initial value. When the experimental time is more than 20 hours, the decline trend of cell metabolic activity state slows down significantly, and the slowed decline trend of cell metabolic activity state can last for more than 60 hours. Among them, between 20h and 60h, the cell metabolic activity state fluctuated around 50% compared with the initial value. Combined with the time when the expression of target gene (IL-10 / IL-8 / hGluc) increased significantly (about 10h), the change of ccell metabolic activity state would not have a great impact on the expectation of the experimental results, and when the time was more than 20h, the cell metabolic activity state was relatively stable, which was conducive to the production and secretion of cytokines by artificial regulation for a long time.
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Observation and analysis of cell migration
In the later experiments, we realized that the role of cytokines depends on its specific role in the immune signal pathway, such as initiating inflammatory response, recruiting other immune cells, conducting signal transduction and so on. Therefore, we selected interleukin-8 for cell migration experiment.
Interleukin-8 (IL-8) is a chemokine that promotes cell survival and proliferation. As a single agent, IL-8 induces progenitor cell mobilization in primates and mice within a few hours; IL-8 also results in immediate mobilization of mature granulocytes.[2]
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.
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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.
Model-based predictive and control system
i.Switch off kinetics of secreted protein and mRNA
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For parameterizing the light-dependent shut-on and shut-off, cells were first illuminated for 15h, then incubated for 30h in the dark. The measured output is the concentration of IL10-mRNA in a single cell normalized to the basal mRNA expression at different time points. The data shows that the mRNA level begins to decline as soon as the light shuts off. Through fitting it with a one-phase exponential decay function, it’s estimated that its half-life is around 1.5 hours.
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We also perform a switch-off experiment to test the kinetics of IL10(cytokines). Because protein is less dynamic than the mRNA, illumination time was set to 20h, then cells were incubated for 30h in the dark. And the data shows that the protein expression level is still rising after shutting off the light, which has a significant time delay effect.
ii.Multi-intensity time gradient experiment
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Figure 25. System response to a step change in light intensity from 0 to 83.25 uW. Different curves correspond to different intensity (a).Measurement of CCK8 (b).Measurement of mRuby (c).Measurement of hGluc
Since some parameters in our model can be intensity-dependent, We need to fit them under different intensities at different time points. Thus we design a time gradient experiment with five different intensities according to the functional relationship between relevant parameters and light intensity. Through comparing the results of the flow cytometer and ELISA, it is clear that the gene expression level is positively related to light intensity on a time scale. Also, the value of CCK8 is rather constant among all these situations, which means the state of cells is nearly stable. Thus we can use this data to help fit some intensity-dependent parameters in the model.
iii.Regulation test
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In this experiment, 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. Although Our 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.
References
- ↑ J Xiong, H Xiao, Z Zhang (2007). An Experimental Research on Different Detection Conditions between MTT and CCK-8. Acta Laser Biology Sinica.
- ↑ Ruth Seggewiss, ... Donald Orlic, in Handbook of Stem Cells, 2004