iGEM 2019 — Team Nanjing_NFLS

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

# Team:Nanjing NFLS/Notebook

* [Home](https://2019.igem.org/Team%3ANanjing_NFLS)
* [Project](https://2019.igem.org/Team%3ANanjing_NFLS/Description)
  + [Description](https://2019.igem.org/Team%3ANanjing_NFLS/Description)
  + [Background](https://2019.igem.org/Team%3ANanjing_NFLS/Background)
  + [Design](https://2019.igem.org/Team%3ANanjing_NFLS/Design)
  + [Experiment](https://2019.igem.org/Team%3ANanjing_NFLS/Experiment)
  + [Demonstrate](https://2019.igem.org/Team%3ANanjing_NFLS/Demonstrate)
  + [Notebook](https://2019.igem.org/Team%3ANanjing_NFLS/Notebook)
  + [Vision](https://2019.igem.org/Team%3ANanjing_NFLS/Vision)
* [Parts](https://2019.igem.org/Team%3ANanjing_NFLS/Parts)
* [Safety](https://2019.igem.org/Team%3ANanjing_NFLS/Safety)

[![](https://static.igem.org/mediawiki/2019/b/b1/T--Nanjing_NFLS--logo.png)](https://2019.igem.org/Team%3ANanjing_NFLS)

* [Model](https://2019.igem.org/Team%3ANanjing_NFLS/Model)
* [Team](https://2019.igem.org/Team%3ANanjing_NFLS/Team_Members)
  + [Team Members](https://2019.igem.org/Team%3ANanjing_NFLS/Team_Members)
  + [Attributions](https://2019.igem.org/Team%3ANanjing_NFLS/Attributions)
  + [Collaborations](https://2019.igem.org/Team%3ANanjing_NFLS/Collaborations)
* [Human Practices](https://2019.igem.org/Team%3ANanjing_NFLS/Overview)
  + [Overview](https://2019.igem.org/Team%3ANanjing_NFLS/Overview)
  + [Integrated Human Practices](https://2019.igem.org/Team%3ANanjing_NFLS/Integrated_Human_Practices)
  + [Public Engagement](https://2019.igem.org/Team%3ANanjing_NFLS/Public_Engagement)
* [Judging](https://2019.igem.org/Team%3ANanjing_NFLS/Judging)

* [Home](https://2019.igem.org/Team%3ANanjing_NFLS)
* [Project](https://2019.igem.org/Team%3ANanjing_NFLS/Description)
  + [Description](https://2019.igem.org/Team%3ANanjing_NFLS/Description)
  + [Background](https://2019.igem.org/Team%3ANanjing_NFLS/Background)
  + [Design](https://2019.igem.org/Team%3ANanjing_NFLS/Design)
  + [Experiment](https://2019.igem.org/Team%3ANanjing_NFLS/Experiment)
  + [Demonstrate](https://2019.igem.org/Team%3ANanjing_NFLS/Demonstrate)
  + [Notebook](https://2019.igem.org/Team%3ANanjing_NFLS/Notebook)
  + [Vision](https://2019.igem.org/Team%3ANanjing_NFLS/Vision)
* [Parts](https://2019.igem.org/Team%3ANanjing_NFLS/Parts)
* [Safety](https://2019.igem.org/Team%3ANanjing_NFLS/Safety)
* [Model](https://2019.igem.org/Team%3ANanjing_NFLS/Model)
* [Team](https://2019.igem.org/Team%3ANanjing_NFLS/Team_Members)
  + [Team Members](https://2019.igem.org/Team%3ANanjing_NFLS/Team_Members)
  + [Attributions](https://2019.igem.org/Team%3ANanjing_NFLS/Attributions)
  + [Collaborations](https://2019.igem.org/Team%3ANanjing_NFLS/Collaborations)
* [Human Practices](https://2019.igem.org/Team%3ANanjing_NFLS/Overview)
  + [Overview](https://2019.igem.org/Team%3ANanjing_NFLS/Overview)
  + [Integrated Human Practices](https://2019.igem.org/Team%3ANanjing_NFLS/Integrated_Human_Practices)
  + [Public Engagement](https://2019.igem.org/Team%3ANanjing_NFLS/Public_Engagement)
* [Judging](https://2019.igem.org/Team%3ANanjing_NFLS/Judging)

Notebook

* [Notebook](#Notebook)
* [General protocols](#protocols)

**Notebook**

|  |  |
| --- | --- |
| **Date** | **Introduction** |
| 28-May-2019 | Obtained   Plasmid extraction |
| 02-Jun-2019 | Preparation   of DH5α competent cells by CaCl2 method |
| 03-Jun-2019 | 1.   Obtained pcDNA3.1, pcDNA6.2-HBsAg plasmid  2. Double   digestion of the pcDNA6.2-HBsAg plasmid and the hTERT plasmid for ligation |
| 04-Jun-2019 | Transferred   pcDNA6.2-HBsAg and hTERT linker into DH5α |
| 15-Jul-2019 | Performed   double digestion on the pcDNA3.1 plasmid and the Ce-125-GAAA plasmid for   ligation. |
| 16-Jun-2019 | 1.   Transferred pcDNA3.1 and Ce-125-GAAA linker into DH5α  2.   Obtained the pcDNA6.2-hTERT-HBsAg plasmid |
| 18-Jul-2019 | Obtained   the pcDNA3.1-Ce-125-GAAA plasmid |
| 23-Jul-2019 | 1.   Verified that pcDNA3.1-Ce-125-GAAA is successfully connected  2. Initial   verification of the correctness of the pcDNA6.2-hTERT plasmid  3.   Prepared DH5α competent cells (transformation) by CaCl2 method |
| 30-Jul-2019 | Double-cleared   the pcDNA6.2-hTERT-HbsAg plasmid and the miR-70 and miR-95 plasmids for   ligation |
| 13-Aug-2019 | Transferred   pcDNA6.2-hTERT-HBsAg to miR-70, miR95 linker into DH5α |
| 15-Aug-2019 | Double-digested   the pcDNA6.2-hTERT-HbsAg plasmid and the miR-125 plasmid for ligation |
| 16-Aug-2019 | Prepared   DH5α competent cells (transformation) by CaCl2 method |
| 18-Aug-2019 | Double-clearing   the pcDNA3.1 plasmid and the Ce-70-GACT and Ce-95-tctc plasmids for ligation |
| 19-Aug-2019 | Transferred   pcDNA3.1 and Ce-70-gact, Ce-95-tctc linker into DH5α |
| 21-Aug-2019 | 1.   Obtained pcDNA3.1-Ce-70-gact, Ce-95-tctc plasmid  2.   Verified that pcDNA3.1-Ce-70-gact and Ce-95-tctc are successfully connected |
| 27-Sep-2019 | 1.   Resuscitated 293T cells  2. Resuscitated   HepG2 cells |
| 30-Sep-2019 | 1. Passed   the passage of the overgrown HepG2 cells.  2. Passed   the passage of the overgrown 293T cells. |
| 02-Oct-2019 | 1.Passed the passage of the overgrown   HepG2 cells into the large bottle.  2. Passed   the passage of the overgrown HepG2 cells. |
| 05-Oct-2019 | 1.Transfected PGL3-Basic, PGL3-Hulc,   PGL3-hTERT, and PGL3-Control plasmids.  2.   Transfected the PGL3 series plasmids |
| 07-Oct-2019 | Detection   of PGL3-Basic, PGL3-Hulc, PGL3-hTERT, PGL3-Control transfection plasmids |

**General protocols**

**Cell cryopreservation**

1. Cryopreservation occurs when the cell density is as long as the cell bottle exceeds 80%.

2. The supernatant was discarded, and 1 mL of trypsin was added to gently shake the trypsin to infiltrate the cell bottle and discard it, and placed at 37 ° C for 1 min.

3. Under the microscope, when the cell morphology became round, it showed complete digestion.

4. Add 1 mL of medium to stop digestion (this step should be done quickly to avoid overdigestion).

5. The cells in the flask were aspirated into a 15 ml centrifuge tube at 1000 rpm for 5 min.

6. Discard the supernatant and add 6 mL of cryopreservation solution (90% complete medium + 10% DMSO)

7. Equally divided into 6 frozen tubes.

8. Quickly place in a freezer box and store at -80 °C.

**Plasmid extraction**

1. Add 250 μl of Buffer P1 containing RNase A to the cell pellet, mix well, and suspend the cell pellet.

2. Add 250 μl Buffer P2, gently mix upside down 4-6 times, mix well, this step can not exceed 5 minutes.

3. Add 350 μl Buffer N3, gently mix upside down 8-10 times, and mix well.

4. Centrifuge at 12,000 rpm for 10 minutes (precipitation was incomplete and centrifuged for another 10 minutes), transfer the supernatant to the adsorption column, centrifuge at 12,000 rpm for 1 minute, and discard the waste.

5. Add 600 μl Buffer PW to the column and centrifuge at 12,000 rpm for 1 minute to remove the waste.

6. Repeat the above steps.

7. Open for 2 minutes.

8. Place in an oven at 55 ° C for 20 minutes.

9. Place the column in a new centrifuge tube and add 50 μl of preheated ddH2O to the middle of the membrane for 2 minutes at room temperature and 1 minute at 12,000 rpm. The resulting solution was re-added to the adsorption column, allowed to stand at room temperature for 2 minutes, and centrifuged at 12,000 rpm for 1 minute.

**PGL3 series Plasmid transfection**

1. Plate the cells one day prior to transfection and switch to antibiotic-free 10% FBS DMEM medium and place 1X105 per well in 24-well plates. According to PGL3-Basic, PGL3-Hulc, PGL3-hTERT, PGL3-Control four groups, each group has four duplicate wells, totaling 16 wells.

2. Dilute Lipofectamine 3000 reagent in opti-MEM medium, 1.5 μL Lipofectamine 3000 per tube. Therefore, the medium 25\*38 = 950 μl and Lipofectamine 3000 1.5\*38 = 57 μl of a total of 1007 μl of the solution were uniformly mixed.

3. Dilute P3000 reagent in opti-MEM medium, prepare according to 2μl/μg plasmid, and mix each group with pRL-tk plasmid in a 10:1 relationship, ie 450ng+45ng/well, to ensure the total amount is 500 ng / hole. Therefore, there are the following systems:

(1) PGL3-Basic: medium 25\*8=200 μl Basic plasmid 17 μl pRL-tk plasmid 1 μl P3000 1\*8=8 μl

(2) For PGL3-Hulc: medium 25\*8=200 μl hulc plasmid 11 μl pRL-tk plasmid 1 μl P3000 1\*8=8 μl

(3) For PGL3-hTERT: medium 25\*8=200 μl hTERT plasmid 12 μl pRL-tk plasmid 1 μl P3000 1\*8=8 μl

(4) For PGL3-control: medium 25\*8=200 μl Control plasmid 5.7 μl pRL-tk plasmid 1 μl P3000 1\*8=8 μl

4. Mix the solutions in steps 2 and 3 uniformly (200 μl plus 200 μl) for 15 minutes, then add 50 μl per well to the 950 μl opti-MEM medium/ The wells were incubated in a 24-well plate in a 37 ° C 5% CO 2 incubator.

5. After 6H, switch to 10% DMEM medium and continue to culture, and test after 36h.

**Preparation of DH5α competent state (transformation)**

1. Take 200 μl of the strain stored at -20 °C and add it to 20 ml of LB medium, and incubate at 37 °C, 220 r/min shaker overnight.

2. The next day, 200 μl of the overnight culture solution was transferred to 20 ml LB, and cultured at 37 °C, 220 r/min for 2 h.

3. Pipette 1.5 ml into the centrifuge tube (8 in total) and centrifuge at 5800 rpm for 5 min at 4 °C.

4. Discard the supernatant in the clean bench, collect the bacterial pellet, add 600 μl of pre-cooled 0.1 mol/L CaCl 2 solution, and gently pipette to uniformly disperse the cells, and ice bath for 30 min.

5. Centrifuge at 5,800 rpm for 5 min at 4 °C.

6. Discard the supernatant in the clean bench, collect the bacterial pellet, add 100 ul of pre-cooled 0.1 mol/L CaCl2 solution, gently blow to make the cells evenly dispersed, and store at 4 °C for use, a total of 8 sticks.

**Preparation of DH5α competent state (transformation)**

1. Take 200 μl of the strain stored at -20 °C and add it to 20 ml of LB medium, and incubate at 37 °C, 220 r/min shaker overnight.

2. The next day, 200 μl of the overnight culture solution was transferred to 20 ml LB, and cultured at 37 °C, 220 r/min for 2 h.

3. Pipette 1.5 ml into the centrifuge tube (8 in total) and centrifuge at 5800 rpm for 5 min at 4 °C.

4. Discard the supernatant in the clean bench, collect the bacterial pellet, add 600 μl of pre-cooled 0.1 mol/L CaCl 2 solution, and gently pipette to uniformly disperse the cells, and ice bath for 30 min.

5. Centrifuge at 5,800 rpm for 5 min at 4 °C.

6. Discard the supernatant in the clean bench, collect the bacterial pellet, add 100 ul of pre-cooled 0.1 mol/L CaCl2 solution, gently blow to make the cells evenly dispersed, and store at 4 °C for use, a total of 8 sticks.

**pcDNA6.2-hTERT double enzyme digestion**

1. Preparation of 2% nucleic acid electrophoresis gel: Weigh 1g agar, add it to 50mL of 1xTAE, mix and dissolve in microwave oven for about 2min. After a little cooling, 5 uL of golden view dye was added, poured into a rubberized plate, and gelled after about 25 minutes.

2. Double digestion of the vector pcDNA6.2-hTERT fragment with restriction endonucleases SpeI-HF and SacI-HF

**Cell recovery**

1. Take a 15 mL centrifuge tube and add 9 mL of serum-free medium.

2. The cells were removed from the -80 ° C freezer and thawed in a 37 ° C water bath (within 1 min).

3. After wiping with a 75% ethanol cotton ball, the cell cryotube was opened and transferred to a centrifuge tube.

4. Centrifuge for 5 min at 1000 rpm and discard the supernatant.

5. Add 1 mL of complete medium to the centrifuge tube, pipette until it is dispersed into a single cell suspension, transfer to a cell culture flask, and add 3 mL of complete medium.

6. Incubate at 37 ° C, 5% CO2.

7. After 24 h, the medium was changed according to the cell growth state.

**Cell inheritance**

1. When the cell culture density is greater than 80%, passage can be carried out.

2. The medium in the flask was discarded and 1 mL of trypsin was added for rinsing to remove residual medium.

3. Add 1 mL of trypsin, gently shake the trypsin to infiltrate the cell bottle, place at 37 °C, digest for 1 min 30 s.

4. Under the microscope, when the cell morphology became round, it showed complete digestion.

5. Add 2 mL of medium directly to stop digestion (this step should be done quickly to avoid overdigestion).

6. The digested cells were transferred to a T75 flask and 9 mL of medium containing 10% FBS was added.

7. Incubate at 37 ° C, 5% CO2.

8. After 24 h, the medium was changed according to the cell growth state.

![](https://static.igem.org/mediawiki/2019/f/f9/T--Nanjing_NFLS--wechat.png)

![](https://static.igem.org/mediawiki/2019/3/30/T--Nanjing_NFLS--ins.png)

![](https://static.igem.org/mediawiki/2019/8/80/T--Nanjing_NFLS--logo-1.png)

![](https://static.igem.org/mediawiki/2019/b/b0/T--Nanjing_NFLS--logo-2.png)


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

# Team:Nanjing NFLS/Experiments

![](https://static.igem.org/mediawiki/2019/3/3c/Igem_mobile_menu_logo.svg)

![](https://static.igem.org/mediawiki/2019/e/ee/2019_team_menu_icon.svg)

[![](https://placehold.it/350x150/ececec/cecece)](https://2019.igem.org/Team%3ANanjing_NFLS)
[HOME](https://2019.igem.org/Team%3ANanjing_NFLS)

TEAM

[Team Members](https://2019.igem.org/Team%3ANanjing_NFLS/Team)
[Collaborations](https://2019.igem.org/Team%3ANanjing_NFLS/Collaborations)

PROJECT

[Description](https://2019.igem.org/Team%3ANanjing_NFLS/Description)
[Design](https://2019.igem.org/Team%3ANanjing_NFLS/Design)
[Experiments](https://2019.igem.org/Team%3ANanjing_NFLS/Experiments)
[Notebook](https://2019.igem.org/Team%3ANanjing_NFLS/Notebook)
[Contribution](https://2019.igem.org/Team%3ANanjing_NFLS/Contribution)
[Results](https://2019.igem.org/Team%3ANanjing_NFLS/Results)
[Demonstrate](https://2019.igem.org/Team%3ANanjing_NFLS/Demonstrate)
[Improve](https://2019.igem.org/Team%3ANanjing_NFLS/Improve)
[Attributions](https://2019.igem.org/Team%3ANanjing_NFLS/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3ANanjing_NFLS/Parts)
[Basic Parts](https://2019.igem.org/Team%3ANanjing_NFLS/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3ANanjing_NFLS/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3ANanjing_NFLS/Part_Collection)

[SAFETY](https://2019.igem.org/Team%3ANanjing_NFLS/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3ANanjing_NFLS/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3ANanjing_NFLS/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3ANanjing_NFLS/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3ANanjing_NFLS/Hardware)
[Measurement](https://2019.igem.org/Team%3ANanjing_NFLS/Measurement)
[Model](https://2019.igem.org/Team%3ANanjing_NFLS/Model)
[Plant](https://2019.igem.org/Team%3ANanjing_NFLS/Plant)
[Software](https://2019.igem.org/Team%3ANanjing_NFLS/Software)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Nanjing_NFLS)

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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

# Team:Nanjing NFLS/Results

![](https://static.igem.org/mediawiki/2019/3/3c/Igem_mobile_menu_logo.svg)

![](https://static.igem.org/mediawiki/2019/e/ee/2019_team_menu_icon.svg)

[![](https://placehold.it/350x150/ececec/cecece)](https://2019.igem.org/Team%3ANanjing_NFLS)
[HOME](https://2019.igem.org/Team%3ANanjing_NFLS)

TEAM

[Team Members](https://2019.igem.org/Team%3ANanjing_NFLS/Team)
[Collaborations](https://2019.igem.org/Team%3ANanjing_NFLS/Collaborations)

PROJECT

[Description](https://2019.igem.org/Team%3ANanjing_NFLS/Description)
[Design](https://2019.igem.org/Team%3ANanjing_NFLS/Design)
[Experiments](https://2019.igem.org/Team%3ANanjing_NFLS/Experiments)
[Notebook](https://2019.igem.org/Team%3ANanjing_NFLS/Notebook)
[Contribution](https://2019.igem.org/Team%3ANanjing_NFLS/Contribution)
[Results](https://2019.igem.org/Team%3ANanjing_NFLS/Results)
[Demonstrate](https://2019.igem.org/Team%3ANanjing_NFLS/Demonstrate)
[Improve](https://2019.igem.org/Team%3ANanjing_NFLS/Improve)
[Attributions](https://2019.igem.org/Team%3ANanjing_NFLS/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3ANanjing_NFLS/Parts)
[Basic Parts](https://2019.igem.org/Team%3ANanjing_NFLS/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3ANanjing_NFLS/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3ANanjing_NFLS/Part_Collection)

[SAFETY](https://2019.igem.org/Team%3ANanjing_NFLS/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3ANanjing_NFLS/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3ANanjing_NFLS/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3ANanjing_NFLS/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3ANanjing_NFLS/Hardware)
[Measurement](https://2019.igem.org/Team%3ANanjing_NFLS/Measurement)
[Model](https://2019.igem.org/Team%3ANanjing_NFLS/Model)
[Plant](https://2019.igem.org/Team%3ANanjing_NFLS/Plant)
[Software](https://2019.igem.org/Team%3ANanjing_NFLS/Software)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Nanjing_NFLS)

# Results

Here you can describe the results of your project and your future plans.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the part's Main Page on the Registry.** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)