Team:SUSTech Shenzhen/Experiments
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Contents
- 1 Experiments
- 2 Construction of Plasmid Vector
- 3 Transfection of cells and construction of experimental cell lines
- 4 Monoclonal screening of transfected cells by flow cytometry
- 5 Expansion and culture of monoclonal cells to construct stable cell lines
- 6 Functionality and Stablity Test for our Cell Lines
- 7 Preliminary illumination experiment
- 8 Mycoplasma Tests
- 9 Repeatef Intensity Gradient Experiments
- 10 Repeated Duration Experiment
- 11 Optimized Light Intensity Experiment
- 12 Optimized Light Intensity Experiment
- 13 Transcription-Translation-Secretion Test
- 14 High Reliability Results Obtained by Repeated Experiments
- 15 Cell State Test
- 16 3D Cell Mass Experiment
- 17 Cell Regulation Experiment
- 18 Cell Migration Experiment
Experiments
Construction of Plasmid Vector
-week1&2
a.5xUAS-hGluc-P2A-mRuby
b.EF1α-GAVPO-Bla
c.5xUAS-IL8-P2A-mRuby
d. 5xUAS-IL10-P2A-mRuby
Main Steps:
1.Amplification of target gene fragments (hGluc, mRuby, IL-8,IL-10) by PCR
2.Enzyme digestion of plasmid vector and target fragment by restriction enzymatic digestion
3.Using Gibson ligation system to connect multiple target genes and plasmid vectors at the same time
4.Transforming the linked plasmid vector into DH5αcompetent cells
5.Screening of Monoclonal Strains Successfully Converted by Ampicillin
6.After successful sequencing, the target strain was amplified to extract plasmid.
Transfection of cells and construction of experimental cell lines
-week3
Main Steps：
1. Put Hela cells into 24-well culture plate ahead of time and make their confluence reach about 80% before transfection.
2.The plasmid was transfected into cells with lipo3000 transfection reagent and screened after 48 hours of culture.
Monoclonal screening of transfected cells by flow cytometry
-week4
Main Steps:
Flow cytometry was used to screen transfected cells according to intracellular mRuby signal intensity.
Expansion and culture of monoclonal cells to construct stable cell lines
-week5
Main Steps:
The selected monoclonal cells were cultured in static for a long time, the medium was changed regularly, the cell status was monitored closely, and the follow-up experiments were carried out when the cell number expanded to a relatively large number.
Medium Composition: DMEM Medium + 10% FBS
Functionality and Stablity Test for our Cell Lines
-week6
Illumination experiments were carried out on cell lines cultured from monoclonal cells using a light system to verify whether the functions of different monoclones were in line with expectations and the differences of expression levels between different cell lines
Main Steps:
1.Functional validation of the isolated monoclonal cells was carried out by using 6-well plate. The cells were illuminated with 460 nm blue light for 48 hours. The intensity of intracellular mRuby and extracellular fluorescence signals were determined by flow cytometry and ELASA.
2.Functional validation experiments were repeated three times.
Preliminary illumination experiment
-week7
Flow cytometry, ELASA and microplate reader were used to characterize the intracellular and extracellular signals of the isolated monoclonal cell lines. Accumulate modeling parameters to prepare for subsequent regulatory experiments
Mycoplasma Tests
-repeated during the whole experiment process
In the process of cell experiment, in order to ensure the normal state of cells in the culture process, in addition to microscopic observation, we also carried out multiple Mycoplasma tests on cells in the whole experiment process, eliminating the adverse effects of this kind of pollution on cell state which is not easy to observe
Main steps:
1. Cell collection
2. Set experiment group, positive control and negative control with different template and primers
3. Set and operate PCR program
Repeatef Intensity Gradient Experiments
-week8
Repeated light intensity gradient experiments were conducted to explore the expression intensity of target genes in cells under different light intensity
Main Steps:
1. Cell preparation（cells transfected with 5xUAS-IL10-P2A-mRuby）
2. Setting the gradient of illumination intensity of 0μW-819.2μW
3.Three repetitive tests
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Repeated Duration Experiment
-week9
Repeated illumination duration experiments to explore the change trend of the expression intensity of the target gene and the target gene expression products in different light periods under the same light intensity
Main Steps:
1. Cell preparation（cells transfected with 5xUAS-IL10-P2A-mRuby）
2. Setting the gradient of illumination intensity of 0h-70h.
3.Three repetitive tests
Optimized Light Intensity Experiment
-week10
Based on the time gradient experiment, we chose 48 hours as the duration of the further intensity gradient experiment. At this time, the expression of the target gene product was more markablely. （by using cells transfected with 5xUAS-IL10-P2A-mRuby)
Optimized Light Intensity Experiment
-week11
On the basis of the light intensity gradient experiment, we choose the corresponding light intensity of about 102 microwatts to carry out further light time gradient experiment. Under this light intensity, the expression intensity of target genes in cells is more markablely. （by using cells transfected with 5xUAS-IL10-P2A-mRuby）
Transcription-Translation-Secretion Test
-during week8 to week13
On the basis of preliminary light intensity and light time gradient experiments, we established a triple description system of transcription-translation-secretion, which used qPCR, flow cytometry and ELISA to characterize the mRNA content, intracellular protein expression and extracellular target gene product secretion
Main Steps:
1.To describe transcription intensity, we operate RT-PCR experiments.
A. Using kits to lyse and extract intracellular RNA
B. reverse transcription with reverse transcription kit to obtain cDNA
C. RT-PCR based on SYBR Green dye
2.To describe the level of protein translation, flow cytometry was used to detect the intracellular signal intensity of mRuby and indirectly reflect the level of translation.
3.To describe the secretion of cytokines or labeled gene products, we directly detected cytokines in cell culture supernatants by ELISA or identified hGlucuc content in supernatants by fluorescein substrates.
High Reliability Results Obtained by Repeated Experiments
-week12&13
The above tests were carried out on the cells of light intensity experiment and light time experiment respectively, and the experiments were repeated many times
Cell State Test
-during week8 to week13
Based on the mature time-light intensity control system and illumination system, we detected the cell status under long-term experiments. Through CCK8 detection method, we mapped the cell status under different experimental types
Main Steps:
1. Cell collection after illumination
2.Co-incubation of CCK8 detection reagent and cells
3.Fluorescence intensity detection
3D Cell Mass Experiment
-week14&15
In order to make the experimental conclusion and the mechanism of cytokine transcription-translation-secretion explored in the experimental process more likely to be put into application, we carried out cell 3D culture and functional verification of cells in mass state
Main Steps:
1. Construction of Cell 3D Culture Environment
2.Continuous culture of cell mass
3.Validation of stability of cell mass
4.Functional verification of cell mass
Cell Regulation Experiment
-week16
According to the experimental parameters accumulated in previous experiments and the results of mathematical modeling, we devote ourselves to the artificial regulation of cytokine production by programming the illumination system in later experiments. Within 150 hours, the expression and secretion of cytokines oscillate in a certain range by changing the light intensity and setting the light interval
Main Steps:
1.Cells preparation(cells transfected with 5xUAS-IL10-P2A-mRuby)
2. Drawing the expected gene expression curve.
3.Setting up corresponding illumination program and illuminating cells
4.At the expected turning point where the target gene expression level changes, the expression of the target gene and the corresponding cell state are characterized by the above-mentioned multiple detection methods.
Cell Migration Experiment
-week17
In later experiments, in order to verify the main function of cytokines, we carried out cell migration experiments. In order to verify the biological function of cytokines, we use cell imaging system and algorithm to continuously track the movement of multiple single cells in migration experiments
Main Steps:
1. Choose IL-8 as the cytokine to be verified.
2.Set up negative control (bilateral culture medium), experimental group (IL-8 and culture medium are located on both sides respectively), positive control (bilateral IL-8).
3.Keep tracking and photographing for 15 hours to obtain data and express the movement rule of cells in migration experiment.
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