iGEM 2019 — Team SUSTech_Shenzhen

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:SUSTech Shenzhen/Notebook

Load Tools

[![Beacon](https://static.igem.org/mediawiki/2019/5/54/T--SUSTech_Shenzhen--beacon.png)](https://2019.igem.org/Team%3ASUSTech_Shenzhen)

Toggle navigation

* [Home](https://2019.igem.org/Team%3ASUSTech_Shenzhen)
* Team

  **Team**

  Learn more about our team.
  [Team Members](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Team)
  [Attributions](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Attributions)
* Project

  **Project**

  Details about *C-hoop*.
  [Description](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Description)
  [Design](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Design)
  [Experiments](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Experiments)
  [Notebook](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Notebook)

  [Results](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Results)
  [Demonstration](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Demonstrate)
  [Safety](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Safety)
* Parts

  **Parts**

  Parts we produce.
  [Overview](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Parts)
  [Contribution](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Contribution)
* Human Practices

  **Human Practices**

  Records the events and communication we have with the society.
  [Standard & Integrated](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Human_Practices)
  [Public Engagement](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Public_Engagement)
  [Collaborations](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Collaborations)
* Model

  **Model**

  Models designed in C-hoop
  [Overview](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Model)
  [Predictive and Control System](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Predictive_and_Control_System)
  [Cell Tracking Analysis](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Cell_Tracking_Algorithm)
* Hardware

  **Hardware**

  Hardware implemented in C-hoop
  [Overview](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Hardware)

  [Automatic Illumination, Culturing, and Collection System (AICCS)](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Hardware?aiccs)
  [Illumination Compartments](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Hardware?Illumination_Compartments)
  [Culturing Compartment](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Hardware?Culturing_compartment)
  [Collection Compartment](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Hardware?Collection_Compartment)
* [Software](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Software)

* [Awards](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Medal_Criteria)

Back to Top

![](https://static.igem.org/mediawiki/2019/4/40/T--SUSTech_Shenzhen--headerSetProject.png)

## Contents

* [1 Cell culture](#Cell_culture)
* [2 Cell counting](#Cell_counting)
* [3 Cell passage](#Cell_passage)
* [4 Cell medium preparation](#Cell_medium_preparation)
* [5 Construction of plasmid vector (molecular clone)](#Construction_of_plasmid_vector_.28molecular_clone.29)
  + [5.1 a.polymerase chain reaction(PCR)](#a.polymerase_chain_reaction.28PCR.29)
    - [5.1.1 Amplification PCR system](#Amplification_PCR_system)
    - [5.1.2 Amplification PCR program](#Amplification_PCR_program)
  + [5.2 b.Restriction endonuclease reaction](#b.Restriction_endonuclease_reaction)
  + [5.3 c.Gibson assembly system(2-3 fragments)](#c.Gibson_assembly_system.282-3_fragments.29)
  + [5.4 d. Preparation of nucleic acid gel](#d._Preparation_of_nucleic_acid_gel)
  + [5.5 e. Nucleic acid gel extraction by kit](#e._Nucleic_acid_gel_extraction_by_kit)
  + [5.6 f.Plasmids transformation](#f.Plasmids_transformation)
* [6 Clone screen](#Clone_screen)
  + [6.1 Colony PCR system](#Colony_PCR_system)
  + [6.2 Colony PCR program](#Colony_PCR_program)
* [7 Cell Transfection by using lipo3000](#Cell_Transfection_by_using_lipo3000)
* [8 Flow cytometry operation](#Flow_cytometry_operation)
* [9 Microplate reader operation](#Microplate_reader_operation)
* [10 Luciferase (hGluc)-substrate fluorescence reaction](#Luciferase_.28hGluc.29-substrate_fluorescence_reaction)
* [11 CCK8 detection reagents-cells fluorescence reaction](#CCK8_detection_reagents-cells_fluorescence_reaction)
* [12 Mycoplasma test](#Mycoplasma_test)
  + [12.1 Mycoplasma test PCR program](#Mycoplasma_test_PCR_program)
* [13 Enzyme-linked immunosorbent ( ELISA) operation(For IL-10、IL-8)](#Enzyme-linked_immunosorbent_.28_ELISA.29_operation.28For_IL-10.E3.80.81IL-8.29)
* [14 RNA extraction(QIAGE RNeasy Mini Kit)](#RNA_extraction.28QIAGE_RNeasy_Mini_Kit.29)
* [15 Plasmids extraction](#Plasmids_extraction)
* [16 Reverse transcription PCR program](#Reverse_transcription_PCR_program)
* [17 Real time polymerase chain reaction (qPCR)](#Real_time_polymerase_chain_reaction_.28qPCR.29)
  + [17.1 qPCR reaction system](#qPCR_reaction_system)
  + [17.2 qPCR program](#qPCR_program)
* [18 3D cell culture dishes preparation](#3D_cell_culture_dishes_preparation)
* [19 3D cell mass preparation](#3D_cell_mass_preparation)
* [20 Cell migration experiment](#Cell_migration_experiment)

## Cell culture

1. Prepare DMEM medium containing 10% FBS

2. Set the cell culture condition as 5% CO2 concentration, 37 ℃.

## Cell counting

1. Mix 10 μ L cell suspension and 10 μ L 4% typany blue solution evenly, and add them to counter star cell counting plate.

2. Count cells with cell counter

## Cell passage

1. Remove the original culture medium with vacuum pump

2. Wash the cell surface with 1 \* PBS solution

3. Digest the Intercellular cell matrix with typsin-EDTA solution so that it does not adhere to the wall.

4. Wash the cells with DMEM medium of twice the volume and collect them into 2ml centrifuge tube.

5. Centrifugate with 200xg speed for 3min, and then remove the supernatant.

6. The cells were resuspended with 1ml medium and passed on in the proportion of 1:10.

## Cell medium preparation

Mix the filtered 10% FBS with DMEM medium, and preheat the medium to 37 ℃ before use.

## Construction of plasmid vector (molecular clone)

### a.polymerase chain reaction(PCR)

#### Amplification PCR system

1.ddH20 15μL

2.10xTaq Buffer with K2SO4 or (NH4)2SO4 2.5μL

3.dNTPs Mix(2mM) 2.5μL

4.Primer-F 1μL

5.Primer-R 1μL

6.MgCl2(25mM) 1.5μL(sometimes integrated in the 10xTaq Buffer)

7.Taq DNA Polymerase(5ng/μL) 0.3μL

8.DNA template 1μL

in total 25μL per sample

#### Amplification PCR program

(1).95°C pre-heating for 2min

for 35 cycles:

(2).95°C dunaturation 15s

(3).55°C for annealing 30s

(4).72°C for elongation in the speed of 1kb/min (5)72°C for fully elongation, 10min

(6) set 4°C for preservation

### b.Restriction endonuclease reaction

1. 1mg plasmid corresponds to 1ul restriction endonuclease

2.10x restriction endonuclease buffer

3.ddH2O to make the reaction system reach 30μL per sample

### c.Gibson assembly system(2-3 fragments)

1.Total amount of fragments：0.02-0.05 pmols\*X μL

2.Gibson Assembly Master Mix(2X) 10μL

3.Deionized H2O 10-X μL

in total 20μL

4. 37°C for 30min, 4°C for preservation

### d. Preparation of nucleic acid gel

1. 1% agarose

3. Heat to complete melting

2. Add 10000x nucleic acid dye (genegreen) after cooling to below 80 ℃.

3. Pour the liquid gel into the mold and select the size of sample hole reasonably

4. cooling to nucleic acid gel solidification

(5. Add 6xDNA loading dye to the sample and mix evenly)

### e. Nucleic acid gel extraction by kit

1. Column balance steps: add 500 μ l of balance solution BL to the adsorption column CA2, centrifuge at 12000rpm for 1min, pour out the waste liquid in the collection pipe, and put the adsorption column back into the collection tube.

2. cut the single purpose DNA strip from the agarose gel and let it weigh in a clean centrifuge tube.

3. Add the equal volume solution PN to the gel mass and place it in a 50 ° C water bath until the gel is completely dissolved.

4. Add the solution obtained in the previous step into an adsorption column CA2, place it at room temperature for 2min, centrifuge it at 12000rpm for 60s, pour out the waste liquid in the collection tube, and put the adsorption column CA2 into the collection tube.

5. Add 600 μ l wash buffer PW to the adsorption columnCA2, centrifuge at 12000rpm for 60s, pour out the waste liquid in the collection tube, and put the adsorption column CA2 into the collection tube.

6. Repeat step 5.

7. Put the adsorption columnCA2 into the recovery header and centrifuge at 12000rpm for 2min.

8. Put the adsorption column CA2 into a clean centrifuge tube, add a proper amount of elution buffer EB to the suspended drop in the middle of the adsorption membrane, and place it at room temperature for 2min. DNA solution was collected by centrifugation at 12000rpm for 2min.

### f.Plasmids transformation

1. Thaw DH5 α cells on ice, 100 μ l per tube

2. Add 10 μ l linkage system to the competent cells and incubate on ice for 30 minutes.

3. Put the conversion system into a 72 ° C water bath and heat shock for 70s.

4. Put the heat stimulated transformation system on ice immediately for 5min.

5. Add 1ml LB medium and culture at 37 ° C for 1-2h

6. Centrifuge 12000xg for 2min, and reserve 100 μ l LB medium

7. Fully resuspend and smear the cells onto the LB solid antibiotic plate.

## Clone screen

1. The monoclonal colonies were selected from the corresponding resistant culture plates and cultured in a centrifuge tube containing 50μL LB medium for a short time.

2. Construct colony PCR system and run the program

### Colony PCR system

1. 2\*mix 5μL

2.Primer-F 0.4μL

3.Primer-R 0.4μL

4. LB medium containing target colony 1μL

5.ddH2O 3.2μL

in total 10μL

### Colony PCR program

(1).95°C pre-heating for 5min

for 25-30 cycles:

(2).95°C denaturation 15s

(3).55°C for annealing 30s

(4).72°C for elongation in the speed of 1kb/min (5)72°C for fully elongation, 10min

(6) set 4°C for preservation

3. based on the UV imaging results of nucleic acid gel, the positive samples were preliminarily determined.

4. Sequence the target plasmid with colony as sample

|  |  |
| --- | --- |
| ![Fig:single colon on antibiotic agar plate ](https://static.igem.org/mediawiki/2019/4/4f/T--SUSTech_Shenzhen--p2Aplasmid-2.png)   Fig:single colon on antibiotic agar plate |  |

## Cell Transfection by using lipo3000

1. Seed cells to be 70%-80% confluent at transfection.

2. Dilute Lipofectamine 3000 reagent in OPTI-MEM medium, mix well

3. Prepare master mix of DNA by diluting DNA in OPTI-MEM medium, then add P3000 reagent, mix well.

4. Add diluted DNA to each tube of diluted Lipofectamine 3000 reagent (1:1 ratio)

5. Incubation in room temperature for 15min.

6. Add DNA-lipid complex to cells.

7. Visualize/analyze transfected cells after 24h.

## Flow cytometry operation

1. Start the flow cytometer and initialize the program.

2. Clean the flow cytometer with ddH2O and NaClO

3. Set corresponding flow detection template

4. For the sample adding operation, the cell suspension needs to be filtered before being added to the flow cytometer.

5. Run "dailyclean" and clean the flow cytometer again with ddH2O and NaClO.

## Microplate reader operation

1. Set the format of enzyme plate, sample position and detection area

2. inlet plate

3. oscillation

4. Select the light-emitting mode (absorbance / chemiluminescence)

5. Export and analyze results

|  |  |
| --- | --- |
| ![Fig:Microplate reader](https://static.igem.org/mediawiki/2019/8/87/T--SUSTech_Shenzhen--p2AELISA-1.jpeg)   Fig:Microplate reader |  |

## Luciferase (hGluc)-substrate fluorescence reaction

1. Add 10 μ l of cell supernatant after light exposure

2. Prepare hglluc substrate (60x) and dilute it with luciferase substrate dilution buffer.

3. Add 60 μ l diluted luciferase substrate to each 96 well standard enzyme hole.

## CCK8 detection reagents-cells fluorescence reaction

1. Wash, digest and resuspend cells

2. Add 10 μ l CCK8 detection regent to each wall of 96 well plate.

3. Add 100 μ L cell suspension to 96 well plate.

4.Incubation at 37 ° C for 2h

5. Use microplate reader to measure 450nm absorbance and analyze data

|  |  |
| --- | --- |
| ![Fig:cck-8 detection reagent with cells](https://static.igem.org/mediawiki/2019/c/cc/T--SUSTech_Shenzhen--cck8.jpg)   Fig:cck-8 detection reagent with cells |  |

## Mycoplasma test

1. Take cell culture supernatant as PCR template

2. Set up the experimental group, positive control (Mycoplasma) and negative control (H2O), and use the specific primers related to Mycoplasma to carry out PCR reaction.

### Mycoplasma test PCR program

(1).98°C pre-heating for 1min

for 30 cycles：

(2).98°C denaturation 10s

(3).58°C for annealing 30s

(4).72°C for elongation in the speed of 1kb/min, 1min

(5)72°C for fully elongation, 10min

(6) set 4°C for preservation

## Enzyme-linked immunosorbent ( ELISA) operation(For IL-10、IL-8)

1.Prepare all reagents and samples to room temperature before use, It os recommended that call samples and standards be assayed in duplicate.

2.Remove unused microplate strips from the plate frame, return them to the foil pouchcontaining the desiccant pack, and reaseal.

3. Plate washing: wash the plate with 1x washing buffer (300 μ L / well) for three times and pat the enzyme plate dry.

4. Sample incubation: add standard sample and sample to be tested, 100 μ L / well, incubation at room temperature for 2h

5. Plate washing: discard the liquid in the well, add 1x washing buffer (300 μ L / well) to wash the plate for three times, and pat the enzyme plate dry.

6.Detection antibody incubation: add the detection antibody pre configured to the working concentration into the enzyme plate, 100 μ L / well, mix well, and incubate at room temperature for 1h.

7. Plate washing: discard the liquid in the hole, add 1x washing buffer (300 μ L / well) to wash the plate for three times, and pat the enzyme plate dry.

8.Add the prepared substrate to the enzyme plate, mix it with 200 μ L / well, and incubate at room temperature in dark for 20min.

9. Termination: Add 20 μ L / well termination solution to the enzyme plate, and gently shake the enzyme plate until the color is uniform.

10.Read 450nm absorbance value within 20min

|  |  |
| --- | --- |
| ![Fig:Enzyme-linked immunosorbent](https://static.igem.org/mediawiki/2019/c/c4/T--SUSTech_Shenzhen--p2AELISA-2.jpg)   Fig:Enzyme-linked immunosorbent |  |

## RNA extraction(QIAGE RNeasy Mini Kit)

1.Cells:harvest a maxium of 1x10^7 cells, as a cell pellet or direct lysis in the vessel.Add the appropriate volumn of Buffer RLT

2.Add 1 volumn of 70% ethanol to the lysate, and mix well by pipetting.

3.Transferup to 700μL of the sample, including any precipitate, to an RNeasy Mini spin colun placed in a 2mL collection tube. Close the lid, and centrifuge for 15s at 8000xg. Discard the flow-through.

4.Add 700μL Buffer RW1 to the RNeasy spin column. Close the lid, and centrifuge for 15s at 8000xg. Discard the flow-through.

5.Add 500μL Buffer RPE to the RNeasy spin column. Close the lid, and centrifuge for 15s at 8000xg. Discard the flow-through.

6. Add 500μL Buffer RPE to the RNeasy spin column. Close the lid, and centrifuge for 2min at 8000xg. Discard the flow-through.

7.Place the RNeasy spin column in a anew 1.5mL collection tube. Add 30-50μL RNase-free water directly to the spin column membrane. Closed the lid, and centrifuge for 1min at 8000xg to elute the RNA.

## Plasmids extraction

1. Pour 1.5 ml overnight grown culture in a microcentrifuge tube. Centrifuge at room temperature (or 4°C) for 60 seconds at 12,000 rpm (or 5,000 rpm for 5 min).

2. Remove the supernatant from the tube completely, leaving the bacterial pellet as dry as possible.

3.Add 500 μl ice cold STE solution. Resuspend the bacterial pellet properly by vortexing or by slow rounds of pipetting. Centrifuge at 4°C for 60 seconds at 12,000 rpm (or 5,000 rpm for 5 min). Remove the supernatant from the tube completely.

4.Add 350 μl of STET solution and resuspend the bacterial pellet properly by vortexing or by slow rounds of pipetting.

5.Add 250μl of freshly prepared solution of lysozyme and mix immediately by vortexing for 5 seconds.

6.place the tube in a boiling water bath for approximately 1 min Centrifuge the tube at maximum speed (14,000 rpm) in a microcentrifuge for 10 min at 4°C or room temperature.

7. Transfer the supernatant containing plasmid promptly in new microcentrifuge tube.

8. Add equal volume of Phenol:Chloroform:Isoamylalcohol (25:24:1) in the supernatant. Mix by vortexing for 10 sec. Centrifuge at maximum speed at 4°C. Transfer the supernatant to fresh microcentrifuge tube.

9. Add equal volume of isopropanol in the supernatant. Mix it by inverting the tube 4 – 6 times. Centrifuge at maximum speed (14,000 rpm) for 30 min at 25°C. Remove the supernatant completely.

10. Add 500 μl of 70% ethanol to the pellet. Close the tube and invert several times. Centrifuge at 14,000 rpm (maximum speed) for 10 min at 25°C. Remove the supernatant completely.

11. Dissolve the pellet in 25 μl sterile double distilled water or TE

## Reverse transcription PCR program

(1). 37°C for 15min

(2). 85°C for 5s

(3). 4°C for preservation

## Real time polymerase chain reaction (qPCR)

### qPCR reaction system

2xqPCR mix 10μL

Forward primer (10 µM stock) 0.4μL

Reverse primer (10 µM stock) 0.4μL

PCR grade water 4.2μl

### qPCR program

(1).Initial denaturation 94°C 2min

For 40 cycles in total

(2).denaturationn 94°C 15s

(3).Annealing, extension, and read fluorescence 60°C 1min

## 3D cell culture dishes preparation

1. Prepare 1.2% agarose gel

2. add 550μL hydrogel into each well of 24-well plate.

3. before the gel is solidified, 3D printing mold is placed in the middle of well to make the dent needed for cell precipitation.

4. after the gel was solidified, the mold was pulled out, adding 200μL per hole DMEN diluted penicillin and streptomycin to incubate 2h for subsequent use.

## 3D cell mass preparation

1. Add 1.5 \* 10 ^ 5 cells to each well.

2. After 24 hours of culture, observe the cell morphology, discard the supernatant, wash the floating cells and adherent cells with 1 x PBS solution, and add DMEM medium for culture again.

3. Repeat the above steps until the cells form a good mass.

## Cell migration experiment

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.

|  |  |
| --- | --- |
| ![Fig:transwell assay operation](https://static.igem.org/mediawiki/2019/c/c2/T--SUSTech_shenzhen--chemotaxis-2.jpg)   Fig:transwell assay operation |  |

|  |  |
| --- | --- |
| ![Fig:group set in cell migration experiment](https://static.igem.org/mediawiki/2019/0/00/T--SUSTech_shenzhen--chemotaxis-3.jpg)   Fig:group set in cell migration experiment |  |


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:SUSTech Shenzhen/Experiments

Load Tools

[![Beacon](https://static.igem.org/mediawiki/2019/5/54/T--SUSTech_Shenzhen--beacon.png)](https://2019.igem.org/Team%3ASUSTech_Shenzhen)

Toggle navigation

* [Home](https://2019.igem.org/Team%3ASUSTech_Shenzhen)
* Team

  **Team**

  Learn more about our team.
  [Team Members](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Team)
  [Attributions](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Attributions)
* Project

  **Project**

  Details about *C-hoop*.
  [Description](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Description)
  [Design](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Design)
  [Experiments](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Experiments)
  [Notebook](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Notebook)

  [Results](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Results)
  [Demonstration](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Demonstrate)
  [Safety](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Safety)
* Parts

  **Parts**

  Parts we produce.
  [Overview](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Parts)
  [Contribution](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Contribution)
* Human Practices

  **Human Practices**

  Records the events and communication we have with the society.
  [Standard & Integrated](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Human_Practices)
  [Public Engagement](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Public_Engagement)
  [Collaborations](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Collaborations)
* Model

  **Model**

  Models designed in C-hoop
  [Overview](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Model)
  [Predictive and Control System](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Predictive_and_Control_System)
  [Cell Tracking Analysis](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Cell_Tracking_Algorithm)
* Hardware

  **Hardware**

  Hardware implemented in C-hoop
  [Overview](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Hardware)

  [Automatic Illumination, Culturing, and Collection System (AICCS)](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Hardware?aiccs)
  [Illumination Compartments](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Hardware?Illumination_Compartments)
  [Culturing Compartment](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Hardware?Culturing_compartment)
  [Collection Compartment](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Hardware?Collection_Compartment)
* [Software](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Software)

* [Awards](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Medal_Criteria)

Back to Top

![](https://static.igem.org/mediawiki/2019/4/40/T--SUSTech_Shenzhen--headerSetProject.png)

## Contents

* [1 Experiments](#Experiments)
* [2 Construction of Plasmid Vector](#Construction_of_Plasmid_Vector)
* [3 Transfection of cells and construction of experimental cell lines](#Transfection_of_cells_and_construction_of_experimental_cell_lines)
* [4 Monoclonal screening of transfected cells by flow cytometry](#Monoclonal_screening_of_transfected_cells_by_flow_cytometry)
* [5 Expansion and culture of monoclonal cells to construct stable cell lines](#Expansion_and_culture_of_monoclonal_cells_to_construct_stable_cell_lines)
* [6 Functionality and Stablity Test for our Cell Lines](#Functionality_and_Stablity_Test_for_our_Cell_Lines)
* [7 Preliminary illumination experiment](#Preliminary_illumination_experiment)
* [8 Mycoplasma Tests](#Mycoplasma_Tests)
* [9 Repeatef Intensity Gradient Experiments](#Repeatef_Intensity_Gradient_Experiments)
* [10 Repeated Duration Experiment](#Repeated_Duration_Experiment)
* [11 Optimized Light Intensity Experiment](#Optimized_Light_Intensity_Experiment)
* [12 Optimized Light Intensity Experiment](#Optimized_Light_Intensity_Experiment_2)
* [13 Transcription-Translation-Secretion Test](#Transcription-Translation-Secretion_Test)
* [14 High Reliability Results Obtained by Repeated Experiments](#High_Reliability_Results_Obtained_by_Repeated_Experiments)
* [15 Cell State Test](#Cell_State_Test)
* [16 3D Cell Mass Experiment](#3D_Cell_Mass_Experiment)
* [17 Cell Regulation Experiment](#Cell_Regulation_Experiment)
* [18 Cell Migration Experiment](#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

|  |  |
| --- | --- |
| ![Fig:Intensity Gradient Experiment](https://static.igem.org/mediawiki/2019/a/a2/T--SUSTech_shenzhen--Blue_light_on_gradient_figure.png)   Fig:Intensity Gradient Experiment |  |

## 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.

|  |  |
| --- | --- |
| ![Fig:Cell Migration](https://static.igem.org/mediawiki/2019/d/d6/T--SUSTech_shenzhen--IL-8_guide_neutrophils_trafficking.png)   Fig:Cell Migration |  |

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:SUSTech Shenzhen/Results

Load Tools

[![Beacon](https://static.igem.org/mediawiki/2019/5/54/T--SUSTech_Shenzhen--beacon.png)](https://2019.igem.org/Team%3ASUSTech_Shenzhen)

Toggle navigation

* [Home](https://2019.igem.org/Team%3ASUSTech_Shenzhen)
* Team

  **Team**

  Learn more about our team.
  [Team Members](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Team)
  [Attributions](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Attributions)
* Project

  **Project**

  Details about *C-hoop*.
  [Description](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Description)
  [Design](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Design)
  [Experiments](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Experiments)
  [Notebook](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Notebook)

  [Results](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Results)
  [Demonstration](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Demonstrate)
  [Safety](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Safety)
* Parts

  **Parts**

  Parts we produce.
  [Overview](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Parts)
  [Contribution](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Contribution)
* Human Practices

  **Human Practices**

  Records the events and communication we have with the society.
  [Standard & Integrated](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Human_Practices)
  [Public Engagement](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Public_Engagement)
  [Collaborations](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Collaborations)
* Model

  **Model**

  Models designed in C-hoop
  [Overview](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Model)
  [Predictive and Control System](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Predictive_and_Control_System)
  [Cell Tracking Analysis](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Cell_Tracking_Algorithm)
* Hardware

  **Hardware**

  Hardware implemented in C-hoop
  [Overview](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Hardware)

  [Automatic Illumination, Culturing, and Collection System (AICCS)](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Hardware?aiccs)
  [Illumination Compartments](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Hardware?Illumination_Compartments)
  [Culturing Compartment](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Hardware?Culturing_compartment)
  [Collection Compartment](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Hardware?Collection_Compartment)
* [Software](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Software)

* [Awards](https://2019.igem.org/Team%3ASUSTech_Shenzhen/Medal_Criteria)

Back to Top

![](https://static.igem.org/mediawiki/2019/4/40/T--SUSTech_Shenzhen--headerSetProject.png)

## 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.

![](https://static.igem.org/mediawiki/2019/d/d3/T--SUSTech_shenzhen--Light_on_system_note.png)

Figure 1. plasmid construction

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.

![](https://static.igem.org/mediawiki/2019/e/e7/T--SUSTech_Shenzhen--result10.jpg)

Figure 2. lentivirus tranfection

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.

![](https://static.igem.org/mediawiki/2019/7/7a/T--SUSTech_Shenzhen--flow.png)

Figure 3. Results of infection

## 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:

![](https://static.igem.org/mediawiki/2019/c/cc/T--SUSTech_shenzhen--Light_gradient_data_example.png)

Figure 4. Intensity gradient

![](https://static.igem.org/mediawiki/2019/d/d8/T--SUSTech_Shenzhen--result02.png)

Figure 5. Illuminating hela-5xUAS-mRuby-P2A-IL10 for 48h with different intensities. a. Measuring the concentration of IL10 by Elisa; b. Measuring red fluorescent with flow cytometer.

 (ii): Relationship between target gene expression and illumination time:

![](https://static.igem.org/mediawiki/2019/2/26/T--SUSTech_shenzhen--Time_gradient_data_example.png)

Figure 6. Time gradient

![](https://static.igem.org/mediawiki/2019/9/94/T--SUSTech_Shenzhen--result04.png)

Figure 7. Illuminating hela-5xUAS-mRuby-P2A-IL10 for 60h under 102.4uw blue light. a. Measuring the concentration of IL10 with Elisa; b. Measuring red fluorescent with flow cytometer.

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

|  |  |
| --- | --- |
| ![](https://static.igem.org/mediawiki/2019/9/96/T--SUSTech_Shenzhen--result05.png)   Figure 8. qPCR test on mRNA of hGluc transcription in 60h light induce experiment | ![](https://static.igem.org/mediawiki/2019/f/fd/T--SUSTech_Shenzhen--result06.png)   Figure 9. qPCR test on mRNA of hGluc transcription in light-dark conversion system |

 (ii): Result of Result of mRuby flow cytometry test for translation characterization and cell number at each time point:
![](https://static.igem.org/mediawiki/2019/9/9d/T--SUSTech_Shenzhen--result07.png)

Figure 10. flow cytometry test on mRuby translation

 (iii) Result of hGluc chemiluminescence value on secretion characterization
![](https://static.igem.org/mediawiki/2019/c/c0/T--SUSTech_Shenzhen--result09.png)

Figure 11. chemiluminescence test on hGluc secretion

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.

|  |  |
| --- | --- |
| ![Fig:3D printed mould](https://static.igem.org/mediawiki/2019/5/50/T--SUSTech_shenzhen--WTQ7.png)   Figure 12. 3D printed mould |  |

|  |  |
| --- | --- |
| ![Fig:3D printed pressed 24-well plate with antibiotic incubation](https://static.igem.org/mediawiki/2019/2/22/T--SUSTech_shenzhen--WTQ4.png)   Figure 13. 3D printed pressed 24-well plate with antibiotic incubation |  |

|  |  |
| --- | --- |
| ![Fig:3D cell mass in minipit](https://static.igem.org/mediawiki/2019/2/20/T--SUSTech_shenzhen--WTQ9.png)   Figure 14. 3D cell mass in minipit |  |

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.

|  |  |
| --- | --- |
| ![Fig:two cell type in the flow cytometry analysis](https://static.igem.org/mediawiki/2019/8/80/T--SUSTech_shenzhen--WTQ1.png)   Figure 15. two cell type in the flow cytometry analysis |  |

|  |  |
| --- | --- |
| ![Fig:3D cell mass stability test](https://static.igem.org/mediawiki/2019/3/38/T--SUSTech_shenzhen--WTQ10.png)   Figure 16. 3D cell mass stability test |  |

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.

|  |  |
| --- | --- |
| ![Fig:intracellular signal test-mRuby intensity test by flow cytometry](https://static.igem.org/mediawiki/2019/4/4f/T--SUSTech_shenzhen--WTQ12.png)   Figure 17. intracellular signal test-mRuby intensity test by flow cytometry |  |

|  |  |
| --- | --- |
| ![Fig:extracellular signal test--fluorescence intensiyt of hGluc-substrate reaction tested by microplate reader](https://static.igem.org/mediawiki/2019/3/37/T--SUSTech_shenzhen--WTQ3.png)   Figure 18. extracellular signal test--fluorescence intensiyt of hGluc-substrate reaction tested by microplate reader |  |

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.

|  |  |
| --- | --- |
| ![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 ](https://static.igem.org/mediawiki/2019/c/c8/T--SUSTech_shenzhen--WTQ11.png)   Figure 19. comparison of intracellular signal with extracellular signal, both the two kinds of the sinals reach their summit at the light power of 89.6μW |  |

## 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]](#cite_note-ref_2-1)

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.

|  |  |
| --- | --- |
| ![Fig:CCK-8 cell state determination](https://static.igem.org/mediawiki/2019/4/4e/T--SUSTech_shenzhen--HGLUC%2870%29_cck8_%28no_adhesion%29.jpg)   Figure 20. CCK-8 cell state determination |  |

## 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]](#cite_note-ref_1-2)

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.

|  |  |
| --- | --- |
| ![](https://static.igem.org/mediawiki/2019/3/3f/T--SUSTech_shenzhen--chemotaxis-1.jpg)   Figure 21. transwell assay sketch map |  |

|  |  |
| --- | --- |
| ![](https://static.igem.org/mediawiki/2019/5/51/T--SUSTech_shenzhen--chemotaxis-4.jpg)   Figure 22. transwell assay plate |  |

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

|  |  |
| --- | --- |
| ![Figure 1. Switch-off kinetics of IL10 mRNA](https://static.igem.org/mediawiki/2019/7/7c/T--SUSTech_shenzhen--ZZ-figure2.png)   Figure 23. Switch-off kinetics of IL10 mRNA |  |

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.

|  |  |
| --- | --- |
| ![Figure 2. Switch-off kinetics of IL10](https://static.igem.org/mediawiki/2019/b/ba/T--SUSTech_shenzhen--ZZ-figure1.png)   Figure 24. Switch-off kinetics of IL10 |  |

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

|  |  |  |
| --- | --- | --- |
| ![](https://static.igem.org/mediawiki/2019/e/e5/T--SUSTech_Shenzhen--multi-cck8.png) | ![](https://static.igem.org/mediawiki/2019/4/45/T--SUSTech_Shenzhen--multi-flow.png) | ![](https://static.igem.org/mediawiki/2019/e/ed/T--SUSTech_Shenzhen--secretion.png) |

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

|  |  |
| --- | --- |
| ![Figure 4. Regulation test](https://static.igem.org/mediawiki/2019/8/89/T--SUSTech_Shenzhen--rgt.png)   Figure 26. Regulation test |  |

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

1. [↑](#cite_ref-ref_2_1-0) J Xiong, H Xiao, Z Zhang (2007). An Experimental Research on Different Detection Conditions between MTT and CCK-8. Acta Laser Biology Sinica.
2. [↑](#cite_ref-ref_1_2-0)  Ruth Seggewiss, ... Donald Orlic, in Handbook of Stem Cells, 2004

Results

---

[Result on construction of LightON system in HeLa cells](#resultLO)
[Result on characterization of light-induced gene expression in Hela cells](#resultCLI)

[1.Determination of optimal illumination conditions](#resultCLI1)
[2. Characterization of the whole expression process at different levels (transcription, translation, and secretion)](#resultCLI2)
[Result of Functionality Test in 3D-culture cells](#result3D)
[Determination of cell activity](#detCellAct)
[Observation and analysis of cell migration](#obsAna)
[Model-based predictive and control system](#mbpcs)

[i.Switch off kinetics of secreted protein and mRNA](#mbpcsi)
[ii.Multi-intensity time gradient experiment](#mbpcsii)
[iii.Regulation test](#mbpcsiii)
[References](#References)