iGEM 2019 — Team CSU_CHINA

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# Team:CSU CHINA/Notebook

[![logo](https://static.igem.org/mediawiki/2019/c/ca/T--CSU_CHINA--LOGOWITHWORDS.png)](/Team%3ACSU_CHINA)

* [SPECIAL PAGE](/Team%3ACSU_CHINA/Special_Page)
* [HOME](/Team%3ACSU_CHINA)
* PROJECT

  [DESCRIPTION](/Team%3ACSU_CHINA/Description)
  [DESIGN](/Team%3ACSU_CHINA/Design)
  [DEMONSTRATION](/Team%3ACSU_CHINA/Demonstrate)
* PARTS

  [BASIC PARTS](/Team%3ACSU_CHINA/Basic_Part)
  [COMPOSITE PARTS](/Team%3ACSU_CHINA/Composite_Part)
  [IMPROVED PART](/Team%3ACSU_CHINA/Part_Collection)
* [MODELING](/Team%3ACSU_CHINA/Model)
* HUMAN PRACTICES

  [INTERGRATED](/Team%3ACSU_CHINA/Human_Practices)
  [PUBLIC ENGAGEMENT](/Team%3ACSU_CHINA/Public_Engagement)
* [SAFETY](/Team%3ACSU_CHINA/Safety)
* LAB

  [LABTASK](/Team%3ACSU_CHINA/LabTasks)
  [NOTEBOOK](/Team%3ACSU_CHINA/Notebook)
  [PROTOCALS](/Team%3ACSU_CHINA/Protocols)
* TEAM

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* [JUDGING](/Team%3ACSU_CHINA/Judging)

![](https://static.igem.org/mediawiki/2019/8/8a/T--CSU_CHINA--SGoldMedal.png)

# loading……

Notebook

≣

![](https://static.igem.org/mediawiki/2019/d/de/T--CSU_CHINA--TimeLineS.jpg)

| 2019 | | | May | | | |
| --- | --- | --- | --- | --- | --- | --- |
| SUN | MON | TUE | WED | THU | FRI | SAT |

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# NOTEBOOK

![](https://static.igem.org/mediawiki/2019/d/de/T--CSU_CHINA--TimeLineS.jpg)

## May

### Main tasks

#### May 13-25

Screening TNBC specific Transcription factors (TFs)

#### May 27-29

Screening of low-expressed miRNA in TNBC

## May 13

We screened for high-expressed genes in tumor groups in all invasive breast cancer from GEPIA database. Then, 245 genes is selected initially.
86 normal breast transcriptome samples and 303 tumor transcriptome samples were downloaded from TCGA (The Cancer Genome Atlas) database. The differentially expressed genes were screened by EdgeR algorithm. And all genes were selected in the GEPIA results set (245).

## May 16

We used the GEPIA database to test the most specific differential-expressed gene in invasive breast cancer, and found out that 11 overlaps with the previously selected 245.
Following the priority order for transcription factor identification, 9 transcription factors were identified.

## May 21

Today we designed the primer of 9 TFs for PCR, and verified the expression of 9 selected TFs with qPCR.

## May 22

Through RT reaction of mRNA and qPCR towards the cDNA, we got the results of qPCR.
According to the expression in TNBC cell lines and normal cell lines, We chose ESR1 and GATA3 to design synthetic promoters.

## May 25

We checked the potential specific miRNA in TNBC cells from articles and verified from TGCA database, then we purchased the primers of these miRNAs and amplified them
The miRNAs we chose: miR-148b , miR-591, miR-125b, miR-34c-3p, miR-26a/26b, miR-216b, miR-101, miR-340, miR-409-3p, miR-489, miR-141, miR146a, miR-542-3p, miR-135a

## May 27

#### 1.We performed reverse transcription on miRNA and got these cDNA

Combine 3μl of 5X RT Primer and 5 μl of double-stranded template in a reaction tube.
Mix thoroughly, then centrifuge briefly to collect the contents at the bottom of the tube.
Incubate at 85℃ for 5min
Incubate at 60℃ for 5min, then place on ice
Add 7μl of RT Reaction Mix to each reaction tube. (details from “Preparing for RT Reaction Mix” showed above)
Centrifuge briefly to collect the contents at the bottom of the tubes

| Step | Temperature | Time |
| --- | --- | --- |
| Reverse transcription | 16℃ | 30min |
| 42℃ | 30min |
| Stop reaction | 85℃ | 5min |
| Hold | 4℃ | Hold |

#### 2.Then we utilized these cDNA for qPCR

|  |  |
| --- | --- |
| SYBR | 5μl |
| F Primer | 0.2μl |
| R Primer | 0.2μl |
| ddH2O | 2.6μl |
| cDNA | 2μl |
| Total | 10μl |
| **Then put them for qPCR machine** | |
| 95℃ for 10min |  |
| 95℃ for 15s | 40 cycles |
| 60℃ for 60s |
| 4℃ reserved |  |

#### 3.Result

![](https://static.igem.org/mediawiki/2019/1/1e/T--CSU_CHINA--LabT2P1F1.png)

## May 29

We chose the specifically high-expressed miRNA: miR-148b and miR-141 for qPCR, and the result was showed below.

![](https://static.igem.org/mediawiki/2019/d/d6/T--CSU_CHINA--LabT2P2F2.png)

## June

### Main Tasks

#### June 3-10

Construction and examination of Binding Sites

## June 3

We purchased the service of synthesizing the whole sequence for binding sites of miR-141 and miR-148b(then got the miR-141-BS and miR-148b-BS) from Sangon Biotech.

| Items | Sequence ( 5‘→3’) | Length |
| --- | --- | --- |
| miR-141-BS | ATTGTGACTAGACCATTTCTAATGCCATTGTGACTAGACCATTTCTAATGCCATTGTGACTAGACCATTTCTAATGCCATTGTGACTAGACCATTTCTAATGGCAA | 106 |
| miR-148b-BS | ACAAAGTTCTGTGATGCACTGAATGCCACAAAGTTCTGTGATGCACTGAATGCCACAAAGTTCTGTGATGCACTGAATGCCACAAAGTTCTGTGATGCACTGAATGGCAA | 110 |

## June 6

#### 1. We took the synthetic sequence(miR-141-BS and miR-148b-BS) for PCR amplification

| Reagent | Volume(μl) |
| --- | --- |
| 5X Buffer | 10 |
| DMSO | 4 |
| dNTP | 4 |
| Forward Primer | 2 |
| Reverse Primer | 2 |
| Targeted DNA fragment | 1 |
| phusion | 1 |
| ddH2O | 26 |
| In Total | 50 |
| 95℃ for 10min |  |
| 95℃ for 15s | n cycles |
| n℃ for 60s |  |
| 4℃ reserved |  |

#### 2. Then, we checked the result of PCR through agarose gel electrophoresis（1% agarose gel；110V for 30min）

#### 3. We cut the fragments of gel and performed purification

| PCR production purification | |
| --- | --- |
| PE Buffer | 750μl/per column |
| PB Buffer | 50μl |
| Elution Buffer | 30μl |
| Gel recycling |  |
| --- | --- |
| PE Buffer | 600μl |
| EB Buffer | 50μl |

#### Finally, we got the amplified products of miR-141-BS and miR-148b-BS and made them stored at -20℃

## June 7

#### 1.We digested the two binding sites with the same incision enzyme with the vector (LSBr5and3), then we connected BS and vector together.

|  |  |  |
| --- | --- | --- |
| **Digestion** | DNA | Vector |
| In Total | 10 | 15 |
| Kpn11 | 1 | 1 |
| Xho1 | 1 | 1 |
| 10XBuffer | 2 | 2 |
| ddH2O | 6 | 11 |
| **Connection** | | Volume |
| Fragment | | 3μl |
| Vector | | 10μl |
| 10XBuffer | | 2μl |
| T4 DNA Ligase | | 3μl |
| ddH2O | | 2μl |
| Total | | 20μl |

#### 2.We got the recombined plasmids to translate them to the competent E.coli cells

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| PGL4.22-Promoter | 0ng | 5 ng | 10 ng | 20 ng |
| β-Gal(line lacZ) | 10ng | 10 ng | 10 ng | 10 ng |
| Empty | 90 ng | 85 ng | 80 ng | 70 ng |
| The total plasmid DNA content of each hole (12 holes in total) in the 24-well plate was 100ng.According to the Lipomax: DNA = 3 mu l;The 100ng ratio is configured with the following optin mixture | | | | |
| +25μl Optin-DNA | Incubate 5min | | | |
| +25μl Optin-Lipomax | incubate5min | | | |
| Blend, then incubate for 20min（the system has a total of 50μl） | | | | |

#### 3.We cultured the bacteria cells with concussion for 12h

## June 8

We transferred the bacteria cells to the solid LB medium with Amp+

|  |  |
| --- | --- |
| Solid LB medium with Amp+ | |
| LB medium （liquid） | 3.96ml |
| Amp | 4μl |

## June 9

We picked up the AmpR+ cells and put miRNA mimics of miR-141 and miR-148b to the liquid medium

## June 10

We took these cells for FACS gauging and observed the rate of different colors expressed by cells

## July

### Main Tasks

#### July 13-24

Construction of Sponge and gauging efficiency of Sponge

#### July 25-30

Construction of Synthetic Promoter and gauging efficiency of promoter (Partly, the rest was done in August)

## July 13

We purchased the primer of sponge of two miRNA: miR-141 and miR-148b

| Items | Sequence(5’→3’) |
| --- | --- |
| F-miR-141-sponge | CGTCTCCAGCCGTAGAAGGA |
| R-miR-141-sponge | CGTCTCCCGAGCATCTTCCT |
| F-miR-148b-sponge | CGTCTCCAGCCACAAAGTTG |
| R-miR-148b-sponge | CGTCTCCCGAGTCAGTGCAT |

## July 15

We got the primer and took up for PCR amplification

| Reagent | Volume(μl) |
| --- | --- |
| 5X Buffer | 10 |
| DMSO | 4 |
| dNTP | 4 |
| Forward Primer | 2 |
| Reverse Primer | 2 |
| Targeted DNA fragment | 1 |
| phusion | 1 |
| ddH2O | 26 |
| In Total | 50 |

Then put the reaction system to the PCR machine

| Cycle | Temperature | Time |
| --- | --- | --- |
|  | 94℃ | 5min |
| 10 cycles Add 0.5℃ after per cycle | 94℃ | 30s |
| 55℃ | 30s |
| 72℃ | 30s |
| 25 cycles | 94℃ | 30s |
| 60℃ | 30s |
| 72℃ | 30s |
|  | 72℃ | 7min |
|  | 4℃ | reserved |

We took the products for agarose gel electrophoresis

We cut the fragments of gel and performed purification

Finally, we got the amplified products of Sponge-miR-141 and Sponge-miR-148b and stored at -20℃

## July 16

We chose to construct the plasmid pEGFP-N1-sponge141\_148b. Through double digestion towards the plasmid and the two Sponge, we made them connected and translated them to the competent E.coli cells

## July 17

We gauged the efficiency of Sponge expression through luciferase assay

## July 25

We designed the primer of the three synthetic promoters used for three Modules

|  |  |
| --- | --- |
| F-ESR1-EcoRI | CCGGAATTCATGACCATGACCCTCCAC |
| R-ESR1-BamHI | CGCGGATCCTCAGACCGTGGCAGGGAA |
| F-GATA3-EcoRI | CCGGAATTCATGGAGGTGACGGCGGAC |
| R-GATA3-BamHI | CGCGGATCCCTAACCCATGGCGGTGAC |
| R-G8p-XhoI | CCGCTCGAGTTTACCAACAGTACCGGA |
| R-lateADEp-XhoI | CCGCTCGAGAGAGTGAGGACGAACGCC |
| F-G8p-SacI | CGAGCTCCGATAGGTACCGAGTTTC |
| F-G8p-KpnI | CGGGGTACCCGATAGGTACCGAGTTTC |

## July 28

We designed the structure of s(GATA3)p for Module1

Plasmid: PGL4.22-s(GATA3)p

![](https://static.igem.org/mediawiki/2019/4/48/T--CSU_CHINA--LabT5P2F5.1.1.png)

## July 30

We designed the structure of s(GATA3)p for Module1

Plasmid: PGL4.22-s(GATA3)p

![](https://static.igem.org/mediawiki/2019/f/f4/T--CSU_CHINA--LabT5P2F5.2.1.png)

## August

### Main Tasks

#### August 6-20

Golden Gate method for connection

#### August 24-30

Construction of Module1

## August 2

We designed the structure of s(ESR1)p for Module3

Plasmid: pGL4.22-G8p

![](https://static.igem.org/mediawiki/2019/0/09/T--CSU_CHINA--LabT5P2F5.3.1.png)

## August 5

We tested the efficiency of promoters.

Plasmid: pGL4.22-hTERT

![](https://static.igem.org/mediawiki/2019/b/b0/T--CSU_CHINA--LabT5P6F5.6.2.png)

![](https://static.igem.org/mediawiki/2019/f/f4/T--CSU_CHINA--LabT5P2F5.2.1.png)

## August 6

We utilized Golden gate method to connect Module1 and Module2

Plasmid: pEGFP-N1-lacZ

![]( https://static.igem.org/mediawiki/2019/c/c3/T--CSU_CHINA--LabT6P3F6.3.1.png)

## August 8

We constructed the plasmid for Module1

![](https://static.igem.org/mediawiki/2019/9/94/T--CSU_CHINA--LabT6P3F6.3.4.1.png)
![](https://static.igem.org/mediawiki/2019/f/f4/T--CSU_CHINA--LabT5P2F5.2.1.png)
![](https://static.igem.org/mediawiki/2019/8/89/T--CSU_CHINA--LabT6P1F6.1.1.png)
![](https://static.igem.org/mediawiki/2019/5/55/T--CSU_CHINA--LabT6P2F6.2.2.png)

Promoter region: BsmBI RS-s(GATA3)p-BsmBI RS; screening and identification region: BsmBI RS-lacZp-lacZ-BsmBI RS

## August 11

We constructed pLN431-s(GATA3)p

Golden gate method to connect the elements

|  |  |
| --- | --- |
| pLN431 plasmid | 2μl |
| s(GATA3)p | 1μl |
| 10XT4 Ligase Buffer | 2μl |
| 10XBuffer | 2μl |
| BsmBI | 1μl |
| ddH2O | 12l |
| Total | 20μl |

Then, we amplified them by PCR

| Step 1(X10) | |
| --- | --- |
| 37℃ | 5min |
| 16℃ | 10min |
| Step 2 | |
| --- | --- |
| 37℃ | 15min |
| 50℃ | 5min |
| 80℃ | 5min |
| Step 3 | |
| --- | --- |
| 16℃ | reserved |

## August 12

We constructed the plasmid for Module2

![](https://static.igem.org/mediawiki/2019/d/d3/T--CSU_CHINA--LabT6P2F6.2.1.jpg)
![](https://static.igem.org/mediawiki/2019/4/48/T--CSU_CHINA--LabT5P2F5.1.1.png)

## August 16

#### 1. pEGFP-N1-Module2(148b): pEGFP-N1-s(ESR1)p-(Sponge-148b)

|  |  |
| --- | --- |
| Vector | 2μl |
| miR141-Sponge | 1μl |
| s(ESR1)p | 1μl |
| 10XT4 Ligase Buffer | 2μl |
| 10XBuffer | 2μl |
| BsmBI | 1μl |
| ddH2O | 11μl |
| Total | 20μl |

#### 2. pEGFP-N1-Module2(141): pEGFP-N1-s(ESR1)p-(Sponge-141)

|  |  |
| --- | --- |
| Vector | 2μl |
| miR141-Sponge | 1μl |
| s(ESR1)p | 1μl |
| 10XT4 Ligase Buffer | 2μl |
| 10XBuffer | 2μl |
| BsmBI | 1μl |
| ddH2O | 11μl |
| Total | 20μl |

#### 3.PCR amplification

| Step 1(X10) | |
| --- | --- |
| 37℃ | 5min |
| 16℃ | 10min |
| Step 2 | |
| --- | --- |
| 37℃ | 15min |
| 50℃ | 5min |
| 80℃ | 5min |
| Step 3 | |
| --- | --- |
| 16℃ | reserved |

## August 20

We tested the construction of Plasmid and important elements via gel electrophoresis

![](https://static.igem.org/mediawiki/2019/2/2d/T--CSU_CHINA--LabT6P4F6.4.1.png)

## August 24

We got the sequences of elements of Module1

Here is the sequence of GAD

![](https://static.igem.org/mediawiki/2019/1/13/T--CSU_CHINA--LabT7P1F7.1.3.png)

## August 25

We turned to golden gate method to connect all the elements of Module1

|  |  |
| --- | --- |
| pLN431 plasmid | 2μl |
| s(GATA3)p | 1μl |
| 10XT4 Ligase Buffer | 2μl |
| 10XBuffer | 2μl |
| BsmBI | 1μl |
| ddH2O | 12l |
| Total | 20μl |

Then, we amplified them by PCR

| Step 1(X10) | |
| --- | --- |
| 37℃ | 5min |
| 16℃ | 10min |
| Step 2 | |
| --- | --- |
| 37℃ | 15min |
| 50℃ | 5min |
| 80℃ | 5min |
| Step 3 | |
| --- | --- |
| 16℃ | reserved |

![](https://static.igem.org/mediawiki/2019/9/95/T--CSU_CHINA--LabT7P1F7.1.1.png)
![](https://static.igem.org/mediawiki/2019/d/de/T--CSU_CHINA--LabT7P1F7.1.2.png)

## September

### Main tasks

#### September 2-11

Construction of Module3

#### September 13-20

Construction of Module2

#### September 21-29

III. Transfection with Transferrin modified liposome

## September 2

|  |  |
| --- | --- |
| R-G8p-XhoI | CCGCTCGAGTTTACCAACAGTACCGGA |
| R-lateADEp-XhoI | CCGCTCGAGAGAGTGAGGACGAACGCC |
| SF-G8p-SacI | CGAGCTCCGATAGGTACCGAGTTTC |
| F-G8p-KpnI | CGGGGTACCCGATAGGTACCGAGTTTC |

## September 5

Basic parts connecting

![](https://static.igem.org/mediawiki/2019/b/b0/T--CSU_CHINA--LabT9P2F9.2.1.png)

yCD

![](https://static.igem.org/mediawiki/2019/e/e6/T--CSU_CHINA--LabT9P2F9.2.3.png)

## September 9

We constructed pLN431-G8p-yCD-(miR-BS)

![]( https://static.igem.org/mediawiki/2019/9/9d/T--CSU_CHINA--LabT9P2F9.2.4.png)

## September 11

We translated the plasmid to E.coli and transferred them to Amp+ solid medium , then picked the single bacterial colony for sequencing whether the plasmid was constructed correctly

## September 13

We connected the elements of Module2 with Golden gate method and amplified.

1.Basic plasmid: pEGFP-N1-LacZ

![](https://static.igem.org/mediawiki/2019/c/c3/T--CSU_CHINA--LabT6P3F6.3.1.png)

2.Elements of Module2

PEGFP-N1- LacZ-s(ESR1)p Sponge

3.Connection by Golden gate method and constructing the whole Module2

We constructed pEGFP-N1-LacZ-miR148b-sponge; pEGFP-N1-LacZ-miR141-sponge; pEGFP-N1-LacZ-miR101-sponge

4.Amplification of Module2

|  |  |
| --- | --- |
| F-s(ESR1)p-BsmBI | GCCATCGTCTCCACTGAGGTCACGGTGACCT |
| R-s(ESR1)p-BsmBI | CGATTCGTCTCCGGCTAGAGTGAGGACGAA |
| F-miR141spe-EcoRI | CCGGAATTCGCCGTAGAAGGAATGTCA |
| R-miR141spe-BamHI | CGCGGATCCGAGCATCTTCCTTACAGT |
| F-miR148bspe-EcoRI | CCGGAATTCGCCACAAAGTTGTGAAAT |
| R-miR148bspe-BamHI | CGCGGATCCGAGTCAGTGCATTTCA |
| F-s(ESR1)p-KpnI | CGGGGTACCAGGTCACGGTGACCTTCT |
| Sponge-141-BsmBI | CGTCTCCAGCCGTAGAAGGAATGTCACAACCAGATAGACCAACACTGTAAGGAAGATGGATAGGTAGAAGGAATGTCACAACCAGATAGACCAACACTGTAAGGAAGATGGATAGGTAGAAGGAATGTCACAACCAGATAGACCAACACTGTAAGGAAGATGGATAGGTAGAAGGAATGTCACAACCAGATAGACCAACACTGTAAGGAAGATGCTCGGGAGACG |
| Sponge-148b-BsmBI | CGTCTCCAGCCACAAAGTTGTGAAATGCACTGACTAGACAAAGTTGTGAAATGCACTGACTAGACAAAGTTGTGAAATGCACTGACTAGACAAAGTTGTGAAATGCACTGACTAGACAAAGTTGTGAAATGCACTGACTAGACAAAGTTGTGAAATGCACTGACTAGACAAAGTTGTGAAATGCACTGACTAGACAAAGTTGTGAAATGCACTGACTCGGGAGACG |

## September 15

We translated the plasmid to E.coli and transferred them to Amp+ solid medium , then picked the single bacterial colony for sequencing whether the plasmid was constructed correctly

## September 20

We tested when Module1+Module3 to examine whether G8p can be expressed.

![](https://static.igem.org/mediawiki/2019/e/e0/T--CSU_CHINA--LabT9P3F9.3.1.png)

## September 21

We utilized the plasmid of Module1 2 3 for co-transfecting

## September 24

![](https://static.igem.org/mediawiki/2019/5/52/T--CSU_CHINA--LabT11P2F11.2.1.png)

HBL-100 with TF-NC for Day1

![](https://static.igem.org/mediawiki/2019/d/d0/T--CSU_CHINA--LabT11P2F11.2.2.png)

HBL-100 with TF-p53 for Day1

![](https://static.igem.org/mediawiki/2019/8/8f/T--CSU_CHINA--LabT11P2F11.2.3.png)

MDA-MB-231 with TF-NC for Day1

![](https://static.igem.org/mediawiki/2019/b/b9/T--CSU_CHINA--LabT11P2F11.2.4.png)

MDA-MB-231 with TF-p53 for Day1

## September 2

![](https://static.igem.org/mediawiki/2019/0/06/T--CSU_CHINA--LabT11P2F11.2.5.png)

HBL- 100 with TF-NC for Day2

![](https://static.igem.org/mediawiki/2019/7/77/T--CSU_CHINA--LabT11P2F11.2.6.png)

HBL- 100 with TF-p53 for Day2

![](https://static.igem.org/mediawiki/2019/5/5f/T--CSU_CHINA--LabT11P2F11.2.7.png)

MDA-MB-231 with TF-NC for Day2

![](https://static.igem.org/mediawiki/2019/b/b0/T--CSU_CHINA--LabT11P2F11.2.8.png)

MDA-MB-231 with TF-p53 for Day2

![](https://static.igem.org/mediawiki/2019/4/4d/T--CSU_CHINA--DSept25.5.png)

DAPI Staining of MDA-MB-231 with TF-P53 For Day1

![](https://static.igem.org/mediawiki/2019/c/ce/T--CSU_CHINA--DSeptember25.6.png)

DAPI Staining of MDA-MB-231 with TF-P53 For Day2

## October

### Main Tasks

#### October 8-10

DAPI staining

## October 8

We used DAPI staining method to observe the system that transfected to MBA-MD-231 and HBL-100 for one day to test whether it worked.

#### 1.We prepared the two cell lines with transfecting the whole circuit, then operated as followed

1. Wash the media with PBS for three times
2. Incubate with PFA for 30-60 minutes
3. Wash the media with PBS for three times
4. Incubate in a solution of 1µg/ml 4,6-diamidino-2-phenylindole (DAPI) for 30 minutes
5. Count cells under fluorescence microscope

#### 2.Results

![](https://static.igem.org/mediawiki/2019/f/f4/T--CSU_CHINA--NOctober8.1.png)

##### Fig.1 HBL-100 transfected with liposome for Day1

![](https://static.igem.org/mediawiki/2019/5/53/T--CSU_CHINA--NOctober8.2.png)

##### Fig.2 MDA-MB-231 transfected with liposome for Day1

## October 9

We left the same cells as Octo.8 after being transfected for the two days , here were the results.

![](https://static.igem.org/mediawiki/2019/a/a3/T--CSU_CHINA--NOct9.1.png)

##### Fig.3 HBL-100 cell line with DAPI Staining under fluorescent microscope for Day2

![](https://static.igem.org/mediawiki/2019/4/40/T--CSU_CHINA--NOct9.2.png)

##### Fig.4 MDA-MB-231 cell line with DAPI Staining under fluorescent microscope for Day2

## October 10

We left the same cells as Oct.8 after being transfected for the three days , then we observed obvious results differentially in TNBC cells and normal cells.

![](https://static.igem.org/mediawiki/2019/8/86/T--CSU_CHINA--OCT10.1.png)

##### Fig.5 HBL-100 cell line with DAPI Staining under fluorescent microscope for Day 3

![](https://static.igem.org/mediawiki/2019/0/03/T--CSU_CHINA--NOct10.2.png)

##### Fig.6 MDA-MB-231 cell line with DAPI Staining under fluorescent microscope for Day3

Comparing Day2 and Day3, we noticed that the whole system transfected to MDA-MB-231 cells successfully worked and killed TNBC cells efficiently and specifically.


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# Team:CSU CHINA/Experiments

[![logo](https://static.igem.org/mediawiki/2019/c/ca/T--CSU_CHINA--LOGOWITHWORDS.png)](/Team%3ACSU_CHINA)

* [SPECIAL PAGE](/Team%3ACSU_CHINA/Special_Page)
* [HOME](/Team%3ACSU_CHINA)
* PROJECT

  [DESCRIPTION](/Team%3ACSU_CHINA/Description)
  [DESIGN](/Team%3ACSU_CHINA/Design)
  [DEMONSTRATION](/Team%3ACSU_CHINA/Demonstrate)
* PARTS

  [BASIC PARTS](/Team%3ACSU_CHINA/Basic_Part)
  [COMPOSITE PARTS](/Team%3ACSU_CHINA/Composite_Part)
  [IMPROVED PART](/Team%3ACSU_CHINA/Part_Collection)
* [MODELING](/Team%3ACSU_CHINA/Model)
* HUMAN PRACTICES

  [INTERGRATED](/Team%3ACSU_CHINA/Human_Practices)
  [PUBLIC ENGAGEMENT](/Team%3ACSU_CHINA/Public_Engagement)
* [SAFETY](/Team%3ACSU_CHINA/Safety)
* LAB

  [LABTASK](/Team%3ACSU_CHINA/LabTasks)
  [NOTEBOOK](/Team%3ACSU_CHINA/Notebook)
  [PROTOCALS](/Team%3ACSU_CHINA/Protocols)
* TEAM

  [COLLABORATIONS](/Team%3ACSU_CHINA/Collaborations)
  [ATTRIBUTIONS](/Team%3ACSU_CHINA/Attributions)
  [TEAM MEMBERS](/Team%3ACSU_CHINA/Team)
* [JUDGING](/Team%3ACSU_CHINA/Judging)

![](https://static.igem.org/mediawiki/2019/8/8a/T--CSU_CHINA--SGoldMedal.png)

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# 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:CSU CHINA/Results

[![logo](https://static.igem.org/mediawiki/2019/c/ca/T--CSU_CHINA--LOGOWITHWORDS.png)](/Team%3ACSU_CHINA)

* [SPECIAL PAGE](/Team%3ACSU_CHINA/Special_Page)
* [HOME](/Team%3ACSU_CHINA)
* PROJECT

  [DESCRIPTION](/Team%3ACSU_CHINA/Description)
  [DESIGN](/Team%3ACSU_CHINA/Design)
  [DEMONSTRATION](/Team%3ACSU_CHINA/Demonstrate)
* PARTS

  [BASIC PARTS](/Team%3ACSU_CHINA/Basic_Part)
  [COMPOSITE PARTS](/Team%3ACSU_CHINA/Composite_Part)
  [IMPROVED PART](/Team%3ACSU_CHINA/Part_Collection)
* [MODELING](/Team%3ACSU_CHINA/Model)
* HUMAN PRACTICES

  [INTERGRATED](/Team%3ACSU_CHINA/Human_Practices)
  [PUBLIC ENGAGEMENT](/Team%3ACSU_CHINA/Public_Engagement)
* [SAFETY](/Team%3ACSU_CHINA/Safety)
* LAB

  [LABTASK](/Team%3ACSU_CHINA/LabTasks)
  [NOTEBOOK](/Team%3ACSU_CHINA/Notebook)
  [PROTOCALS](/Team%3ACSU_CHINA/Protocols)
* TEAM

  [COLLABORATIONS](/Team%3ACSU_CHINA/Collaborations)
  [ATTRIBUTIONS](/Team%3ACSU_CHINA/Attributions)
  [TEAM MEMBERS](/Team%3ACSU_CHINA/Team)
* [JUDGING](/Team%3ACSU_CHINA/Judging)

![](https://static.igem.org/mediawiki/2019/8/8a/T--CSU_CHINA--SGoldMedal.png)

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# 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)