iGEM 2019 — Team BM-AMU

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MARCH

**2019.3.25-2019.3.31**

Selection of the experimenters.

APRIL

**2019.4.1-2019.4.7**

Discussed and decided the project plan and prepared for the experiment equipment and reagents.

**2019.4.8-2019.4.21**

1. Obtained the sequences of E-Cad and N-Cad, designed the LA and RA homologous arms of E-Cad and the upstream and downstream primers. The sequences of tdTomato and eGFP were also obtained by PCR amplification.
Completed the construction of the homologous recombinant plasmid pBKS-hE-Cad-tdTomato.

2. Introduced homologous recombinant plasmids into DH5-α competent cells, selected 20 single colonies and identified them.

**2019.4.22-2019.4.26**

1. Selected PAM sequence, constructed target plasmids.

2. Thawed and cultured WA09, A549, beas-2b, MCF7.

**2019.4.26-2019.4.30**

Detected the expressing levels of E-Cad & N-Cad in wild type WA09, A549, beas-2b, MCF7.

MAY

**2019.5.1-2019.5.13**

1. Lipofection: imported pBKS-hE-Cad-tdTomato and target plasmids into WA09, A549, BEAS-2b, MCF7.

2. Fluorescence observation, monoclonal selection, PCR amplification and identification for the target sequence.

3. Verified that the knock-in of tdTomato in A549, BEAS-2b, MCF7 failed.

4. Obtained the knock-in cell line of tdTomato in WA09.

**2019.5.14-2019.5.24**

The construction of LG-TetO-IN-hSnail1, pPylZ-tTR-KRAB, pPylZ-hTwist1-ERT2.

**2019.5.14-2019.5.27**

1. Lipofection: imported pBKS-hE-Cad-tdTomato and target plasmids again into A549, BEAS-2b, MCF7.

2. Fluorescence observation, monoclonal selection, PCR amplification and identification for the target sequence.

3. Verified that the knock-in of tdTomato in A549, BEAS-2b, MCF7 failed again.

**2019.5.27-2019.5.31**

Extraction of RNA from WA09-tdTomato cell line.

JUNE

**2019.6.1-2019.6.7**

Proved that tdTomato was inserted into the correct location in genome and cDNA levels.

**2019.6.8-2019.6.21**

Designed the LA and RA homologous arms of N-Cad and the upstream and downstream primers. And the sequences of eGFP were also obtained by PCR amplification.
Completed the construction of the homologous recombinant plasmid pBKS-hNCad-eGFP.

**2019.6.22-2019.6.28**

Selected PAM sequence, constructed target plasmids.

JULY

**2019.6.29-2019.7.15**

Actinomycin treatment and WB analysis of protein samples: actinomycin treated WA09-tdTomato and wild-type control group.

**2019.7.5-2019.7.25**

Introduced homologous recombinant plasmids and target plasmids into DH5-α competent cells, selected 20 single colonies, identified .

Lipofection: imported pBKS-hNCad-eGFP and target plasmid into WA09-tdTomato.

**2019.7.25-2019.7.30**

1. Verified that the knock-in of eGFP in WA09-tdTomato failed

2. Chongqing meetup

AUGUST

**2019.7.31-2019.8.10**

1. introduced homologous recombinant plasmids and target plasmids into DH5-α competent cells, selected 20 single colonies, identified again.

2. Lipofection: imported pBKS-hNCad-eGFP and target plasmid into WA09-tdTomato again.

**2019.8.10-2019.8.17**

1. Completed the CAG promoter strength measurement experiment. Compared the GFP expression under the action of different promoters, including CAG, CMV, EF1a and Ubiquitin in 293 cells by flow cytometry.

2. Preparation for PPT and poster of CCIC.

**2019.8.19-2019.8.24**

Take part in CCIC.

SEPTEMBER

**2019.8.25-2019.9.5**

Verified that the knock-in of eGFP in WA09-tdtomato failed again.

Adjustment for project.

**2019.9.10-2019.9.20**

Treat WA09-tdTomato with TGF-beta.

**2019.9.15-2019.10.2**

Used lentiviral transfection to put LG-TetO-IN-hSnail1，pPylZ-tTR-KRAB and pPylZ-hTwist1-ERT2 into WA09-tdTomato separately, made a test about the relationship between efficiency and time/dosage.

**2019.9.21-2019.10.3**

Compared the efficiency of rtTA, TetR and tetr-krab systems. After cell transfection, treated cells with DOX for 24 hours, recorded and detected relative fluorescence expression by flow cytometry.

**2019.9.20-----now**

Data processing, WIKI writing, presentation preparation.

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)  Third Military Medical University,Gaotanyan St 30, Chongqing 400038, China


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# Team:BM-AMU/Experiments

Team:BM-AMU- 2019.igem.org

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# **Experiments**

#### **Plasmid Construction**

In order to construct a dual fluorescence reporter cell model,
we constructed pBKS-hECad-tdTomato [（BBa\_K3120018）](http://parts.igem.org/Part%3ABBa_K3120018)and pBKS-hNCad-EGFP vector plasmids [（BBa\_K3120017）](http://parts.igem.org/Part%3ABBa_K3120017).
Next, we cloned selected sgRNAs into Px330 vector to knock the fluorescent protein into the target gene by CRISPR/cas9 technology.
These plasmids are detailed in **Figure 1.**

![Experiment](https://static.igem.org/mediawiki/2019/7/79/T--BM-AMU--fig2_plasmid.png)

**Figure1**

pBKS-hECad-tdTomato[（BBa\_K3120018）](http://parts.igem.org/Part%3ABBa_K3120018) : composing of pBluescriptIclI SK vector skeleton, the left segment of
termination codon of E-Cadherin CDS sequence is homologous left arm, and the right segment of termination codon is homologous right arm, and the tdTomato CDS sequence inserted in the middle.

pBKS-hNCad-eGFP[（BBa\_K3120017）](http://parts.igem.org/Part%3ABBa_K3120017) : composing of pBluescriptIclI SK vector skeleton, the left segment of
termination codon of N-Cadherin CDS sequence is homologous left arm, and the right segment of termination codon
is homologous right arm, as well as the inserted eGFP CDS sequence and loxP-Neo-loxP. Neo resistance genes play
a role in the absence of Cre enzyme. Because N-Cadherin does not express in the epithelium, it is impossible to identify
the insertion of eGFP after N-Cadherin by fluorescence, and then to screen
the Neo resistance genes. After successful screening, Gre enzyme was added and loxP-Neo-loxP was removed.

pX330（[BBa\_K3120015](http://parts.igem.org/Part%3ABBa_K3120015),
[BBa\_K3120016](http://parts.igem.org/Part%3ABBa_K3120016)）: composing of U6 promoter, 20 bp long gRNA, CAS9 and AMP resistance genes.
The cas9 protein first binds to gRNA, then forms
a complex structure with DNA under the guidance of PAM sequence, and then cuts the double strands of target DNA.

In order to manipulate the concentration of exogenous factor Dox/Tamoxifen to artificially regulate the expression
of EMT-TFs (Snail1, Twist1) in cells and to obtain EMT cells in different states,
we constructed LC-TetO-IN-hSnail1, pPyIZ-hTwist1-ERT2, pPyIZ-tTR-KRAB plasmids.These plasmids are detailed in Figure 2.

![Experiment](https://static.igem.org/mediawiki/2019/3/31/T--BM-AMU--fig1_plasmid.png)

**Figure2**

pPyIZ-TetR-KRAB[（BBa\_K3120013）](http://parts.igem.org/Part%3ABBa_K3120013): After CAG promoter, TetR and KRAB coding sequences were connected in series, including a nuclear localization signal NLS, and IRES introduced Zeocin resistance gene.

pPyIZ-hTwist1-ERT2 [（BBa\_K3120020）](http://parts.igem.org/Part%3ABBa_K3120020): The TWIST1 CDS region was linked to ERT2 coding sequence by CAG promoter. IRES was introduced into Zeocin resistance gene.

LC-TetO-IN-hSnail1[（BBa\_K3120019）](http://parts.igem.org/Part%3ABBa_K3120019):Seven tandem TRE sequences and CAG promoters constitute TetON promoters. SNAIL1 CDS and IRES in tandem introduces NEO resistance genes. LTR sequences are introduced at the 3'and 5'ends of this part, respectively.

#### **Construction of Monitor**

We designed a knock-in strategy for a reporter cassette that results in a gene fusion facilitating the concordant expression of E-Cadherin and td-Tomato. Briefly, 90% confluent hES cells WA09 (WiCell) were digested into single cells with accutase. Cells were centrifuged and resuspended with the CRISPR construct and reporter plasmid in nucleofection media (Lonza). Cells were electroporated and then cultured on plates in mTESR1 media with ROCK inhibitor. After 48 h, cells were digested and single cell was inoculated into the 96 well plate by FACS sorting. After transfecting the plasmid pBKS-hE-Cadherin-tdTomato and PX330 into WA09 cells for CRISPR/cas9 technology to knock in genes, we selected more than four clones to identify whether the fluorescent protein tdTomato was inserted into the correct site at the genome level. We proved the single-cell clone E1,E2 and E4 was correct, which fluorescent proteins tdTomato knocked into a piece of DNA in the genome that fits our goals.While the E3 is wrong. Next, we validated clone E1（WA09 E1） at genome and cDNA levels, respectively.The result shows that the fluorescent protein tdTomato was inserted at the correct location. To investigate whether the fusion expression of fluorescent protein tdTomato and E-Cadherin affected the degradation of the protein, Actinomycin treatment (18.75umol/mL, The duration of drug treatment is 0hr,3hr,6hr,12hr and 24hr ) was performed. The results of Western blot showed that protein degradation efficiency of fusion expressed protein was consistent with that of E-Cadherin. We also observed the subcellular localization of the cells, and the results showed no difference from those reported in the literature.The above results indicate that tdTomato inserts into the correct site and function normally.

In order to establish the fluorescence model, we need to transfer pBKS-hECad-tdTomato [（BBa\_K3120018）](http://parts.igem.org/Part%3ABBa_K3120018) and pBKS-hN-Cad-eGFP [（BBa\_K3120017）](http://parts.igem.org/Part%3ABBa_K3120017) into cells to function. Lipofectamine 2000 Reagent and Lonza electroporation were used. Fluorescence microscopy showed that our transfection was successful. Next, in order to confirm that the fluorescent protein did knock into the correct site, we selected the cells for monoclonal and flow sorting, extracted RNA, PCR analysis and Western Blot from the selected cells. The results showed that the fluorescent protein and target gene were successfully fused and expressed.

#### **Construction of Controller**

Transfected plasmids pPyIZ-hTwist1-ERT2 [（BBa\_K3120020）](http://parts.igem.org/Part%3ABBa_K3120020) into cells WA09 E1 by Lipofectamine 2000 Reagent.The transfected cells were screened with 10μg/ml zeocin. In order to obtain different states of EMT cells by changing the concentration of Tamoxifen，We set up the concentration gradient and time gradient of Tamoxifen respectively, and detected the mRNA expression of EMT-related genes by qPCR.

In order to obtain a cell line which has stable Tet-on system, lentivirus was used for transfection. LC-TetO-IN-hSnail1 [（BBa\_K3120019）](http://parts.igem.org/Part%3ABBa_K3120019) was constructed and co-transfected with packaging plasmids pSPAX2 and pMD2G respectively. The plasmids were transfected into 293FT cells according to the instructions of Lipofectamine 2000. When obvious cytopathic changes occur (transfection 72 hours), culture supernatant was taken and virus was taken by centrifugation at 6000 r/min.Infecting cells with the resulting lentivirus （MOI=5）.10 μg/ml zeocin was used to screen the transfected cells. Then we transfected pPyIZ-TetR-KRAB( [（BBa\_K3120013）](http://parts.igem.org/Part%3ABBa_K3120013) ). In order to obtain EMT cells in different states by changing DOX treatment，We set up the concentration gradient and time gradient respectively, and detected the mRNA expression of EMT-related genes by qPCR.

#### **Experiments for Bronze and Gold medals**

In the construction of controller, CAG Promoter is used in Tet-on and Tamoxifen induced expression system,
but up to now there is no quantitative data table. In order to know the strength of CAG promoter, we designed experiments
to compare the efficiency of CAG promoter with other common promoters.Detailed design: We constructed four plasmids. In the
pGuide vector skeleton, CAG, CMV, EF1a and Ubiquitin promoters were used to initiate the over-expression of GFP, and the expression
intensity of GFP represented the strength of promoter. After four plasmids were constructed, these plasmids were transfected into
HEK293FT cells by Lipofectamine 2000 Reagent, and GFP was successfully expressed. Samples were collected 24 and 48 hours after
transfection.
Flow cytometry was used to compare the expression of GFP, and finally the strength of these promoters was obtained.

In addition, due to the leakage problem of the classical Tet-on system, we improved the classical Tet-on system and fused
TetR with KRAB. The suppressed gene is expressed when Dox is added. In order to prove that our modification is effective,
we compared efficiency of rtTA, TetR and TetR-KRAB. The specific design is: Improvement Verification Experiment of TetR-KRAB. Equal amount of HEK293FT cells were seeded in a 6-well plate ，3 groups co-transfected with plasmids LC-TetO-GFP and plasmid expressing repressors severally. Flow cytometry was used to detect GFP expression on 5, 7, 9 days after transfection. On the day 9, DOX (1 μg/μL) was added to initiate downstream gene expression, and GFP expression was detected 5 days later. HEK293FT cells were used as blank control at each stage.

#### **FACS-Seq Sample Preparation**

After the successful construction of Monitor and Controller, we obtained EMT cells in different intermediate states by changing the dose and time of Dox/Tamoxifen treatment. Next, cells were sorted into several populations according to their fluorescence intensity. The transcriptome data of each sample was then analyzed using RNA-seq.

#### **Transcriptome sequencing**

###### **The experimental process:**

1) Sample of RNA by oligo T primers into reverse transcription reaction, generating the first cDNA chain;

2) Generate double-stranded cDNA through the joint reaction of RNase H, DNA polymerase and T4 ligase;

3) Double chain cDNA by Tn5 enzyme fragmentation, and cover the RD sequencing on the double end sequence below;

4) Through (3) combined with RD sequences at the ends of the connection attributes (P5, se-quencing primers on both ends of the P7, hand in hand enrichment of PCR amplification;

5) Library mixed for sequencing.

![Experiment](https://static.igem.org/mediawiki/2019/c/c7/T--BM-AMU--extp.png)

###### **The flow chart of Data analysis**

![Experiment](https://static.igem.org/mediawiki/2019/e/e2/T--BM-AMU--extp1.png)

**References:**

1. Bolger AM, Lohse M, Usadel B. Trimmomatic: A flexible trimmer for illumina sequence data.Bioinformatics. 2014;30:2114–20.

2. Kim D, Langmead B, Salzberg SL. HISAT: A fast spliced aligner with low memory requirements. Nature methods. 2015;12:357.

3. Langmead B, Salzberg SL. Fast gapped-read alignment with bowtie 2. Nature methods. 2012;9:357.

4. Frankish A, Diekhans M, Ferreira A-M, Johnson R, Jungreis I, Loveland J, et al. GENCODE reference annotation for the human and mouse genomes. Nucleic acids research. 2018;47:D766–73.

5. Liao Y, Smyth GK, Shi W. FeatureCounts: An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2013;30:923–30.

6. StatQuest. RPKM, FPKM and TPM, clearly explained. RNA-seqblog. 2015.

7. Anders S, Huber W. Differential expression analysis for sequence count data. Genome Biology.2010;11:R106.

8. Cock PJ, Fields CJ, Goto N, Heuer ML, Rice PM. The sanger fASTQ file format for sequences with quality scores, and the solexa/Illumina fASTQ variants. Nucleic acids research. 2009;38:1767–71.

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# Team:BM-AMU/Results

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# **Results**

#### **Construction of monitor**

In order to establish the fluorescence model, we need to transfer pBKS-hE-Cadherin-tdTomato and pBKS-hN-Cadherin-eGFP into cells to function. After verification, our hE-Cadherin-tdTomato cell model was successfully constructed, but the hN-Cadherin-eGFP model was unsuccessful, data not shown. The following data are the main figures in the verification process of the hE-Cadherin-tdTomato model.

![Experiment]( https://static.igem.org/mediawiki/2019/b/b6/T--BM-AMU--fig1_results.jpg)

**Figure 1:hE-Cadherin-tdTomato was constructed successfully.**

After transfecting the hE-Cadherin-tdTomato plasmid into WA09 cells, we selected four clones to identify whether the fluorescent protein tdTomato was inserted into the correct site at the genome level (**Figure 1A and 1B**), we proved the single-cell clone E1,E2 and E4 was correct, which fluorescent proteins tdTomato inserted into a piece of DNA in the genome that fits our goals.While the E3 is wrong.Next, we validated clone E1（WA09 E1） at genome and DNA levels, respectively（**Figure1C**）.The result shows that the fluorescent protein tdTomato was inserted at the correct location. To investigate whether the fusion expression of fluores-cent protein tdTomato and ECAD affected the degradation of the protein, Actinomycin treatment (18.75umol/mL, The duration of drug treatment is 0hr,3hr,6hr,12hr and 24hr ) was performed (**Figure1D**). The results of Western blot showed that protein degradation efficiency of fusion expressed protein was consistent with that of ECAD. We also observed the subcellular localization of the cells, and the results showed no dif-ference from those reported in the literature(**Figure1E**).The above results indicate that tdTomato inserts into the correct site and can function correctly.

#### **Construction of controller**

##### **2.1. TWIST-ERT2&Tamoxifen Induced expression system works well.**

![Experiment]( https://static.igem.org/mediawiki/2019/0/0b/T--BM-AMU--Rest2.jpg)

**Figure 2：Tamoxifen induced expression system works well.**

Transfected plasmids pPyIZ-TetR-KRAB（BBa\_K3120013）and pPyIZ-hTwist1-ERT2 （BBa\_K3120020） into cells WA09 E1 by Lipofectamine 2000 Reagent.The transfected cells were screened with 10μg/ml zeocin.In order to obtain different states of EMT cells by changing the concentration of Tamoxifen，We set up the concentration gradient(figure 2A) and time gradient (figure 2B) of Tamoxifen respectively, and detected the mRNA expression of EMT-related genes by qPCR(figure 2A and 2B). With the increase of Tamoxifen concentration and time, TWIST1 expression level gradually increased, epithelial gene marker E-Cadherin expression level gradually decreased, and mesenchyme gene markers N-Cadherin, ZEB1, Vimentin expression level gradually increased.Changes in expression of these genes suggest that our tamoxifen induction system can induce EMT successfully by leading TWIST1 into nuclear . Figure 2D indicates that the red fluorescence intensity of cells decreases with the increase of Tamoxifen concentration，which at fluorescence level confirm that Tamoxifen induction system was constructed successful.

##### **2.2. TetO-Snail &DOX Induced expression system works well.**

![Experiment]( https://static.igem.org/mediawiki/2019/5/5f/T--BM-AMU--Rest3.jpg)

**Figure 3：DOX-Tet/ON Induced expression system works well.**

In order to obtain a cell line stably expressing TET-ON, lentivirus was used for transfection. Snail-induced expression plasmid(LC-TetO-IN-hSnail1,BBa\_K3120019) was constructed and co-transfected with packaging plasmids pSPAX2 and pMD2G respectively. The plasmid was transfected into 293FT cells according to the instructions of Lipofectamine 2000. When obvious cytopathic changes occur (transfection 72 hours), culture supernatant is taken and virus is taken by centrifugation at 6000 r/min.Infecting cells with the resulting lentivirus （MOI=5）.10 μg/ml zeocin was used to screen the transfected cells.In order to obtain EMT cells in different states by changing the concentration of DOX added，We set up the concentration gradient(Figure 3A) and time gradient(Figure 3B) respectively, and detected the mRNA expression of EMT-related genes by qPCR. With the increase of DOX concentration and time, the expression level of SNAIL1 increased gradually. The expression level of E-Cadherin, an epithelial gene marker, decreased gradually, while that of interstitial gene marker N-Cadherin、ZEB1、Vimentin increased gradually.Changes in expression of these genes suggest that our tamoxifen induction system can induce EMT successfully by controlling SNAIL1 expression.

#### **3. Experiments for Bronze and Gold medals**

![Experiment]( https://static.igem.org/mediawiki/2019/5/50/T--BM-AMU--res_fig4.jpg)

**Figure 4A ：Promoter activity comparison.** The results obtained by FACS are as shown in the Figure 4A（left）, but because the absolute value is arbi-trary unit and can not be directly compared with other systems, fluorescence is measured by Median Fluores-cence Intensity. After treatment, the relative strengths of four promoter devices were obtained. Figure 4A（right） indicates that for each promoter, the 48-hour promoter strength is stronger than the 24-hour sample strength. Among them, CAG promoter activity is relatively weak, while CMV promoter has the strongest 48-hour strength, which is about twice as strong as CAG, while Ubiqutin, EF1a are stronger than CAG in varying degrees.

**Figure 4B-4D:**  Improvement Verification Experiment of TetR-KRAB.Figure 4B-4D show that equal amount of HEK293FT cells were seeded in a 6-well plate and transfected with TetO-GFP. Flow cytometry was used to detect GFP expression on 5, 7, 9 days after transfection. On the day 9, DOX (1 μg/μL) was added to initiate downstream gene expression, and GFP expression was detected 5 days later. HEK293FT cells were used as blank con-trols at each stage. The results of the FACS illustrates that without induction with doxycycline, GFP in the original system is still expressed. However, our improved part prevents the leakage of fluorescent proteins. From the slope of Figure 4B, after adding DOX, the improved part turns on the expression of fluorescent pro-tein faster than the original part.

#### **4. Transcriptome sequencing**

In previous experiments, we hope to induce EMT with more clear and controllable factors. The advantage of this method is to make the process of EMT more delicate and stable. We designed a TetON-SNAIL1 system. The ultimate effect of this system is to achieve SNAIL1 overexpression in our cell model, and then we cap-tured and analyzed the differences of cell fluorescence phenotypes. Therefore, we are interested in the dif-ference of fluorescence phenotype induced by SNAIL1 overexpression compared with the difference of tran-scriptome between EMT subtypes.We infected WA09-E1 with LC-TetO-IN-hSnail1 BBa\_K3120019(Lentivirus envelops，MOI=5). The cells were divided into four groups, named S1, S2, S3 and S4, and the four groups were se-quenced by transcriptome.

![Experiment]( https://static.igem.org/mediawiki/2019/c/c6/T--BM-AMU--part6.jpg)

**Figure 5: Differential expression of EMT-related gene.**

**Figure 5:** We first analyzed the sequencing results of ECAD and EMT related markers. The results showed that the trend of ECAD expression as a flow sorting standard was in line with the experimental facts, and there were relative changes in EMT related markers.

![Experiment]( https://static.igem.org/mediawiki/2019/3/3c/T--BM-AMU--fig6.jpg)

**Figure 6: Bubble Map and Cluster Map of Gene differential expression and pathway changes in EMT.**

**Figure 6:**

**Bubble Map:**  The list of differentially expressed genes among samples was enriched and annotated at the physical process layer of GO database, and the results of the first 20 functional pathways of enrichment sig-nificance were visualized on the Bubble Map. In the figure, the abscissa is Rich Factor, and the percentage of the genes on the table enrichment accounts for the annotated genes; the number of genes on the vertical co-ordinate table enrichment; the number of genes on the point enrichment, and the Qvalue value value of the Yan table, the lower, the more significant. [Refer to the attachment for specific pathway information](https://static.igem.org/mediawiki/2019/d/d8/T--BM-AMU--Res1.zip)

**Cluster map:**  Difference analysis showed that: S1VS.S2, 707 genes up-regulated, 785 genes down-regulated, S2VS.S3, 894 genes up-regulated and 579 genes down-regulated; S3VS.S4, 580 genes up-regulated and 902 genes down-regulated. (The specific changes should be accompanied by hyperlinks). Each point in the graph represents a gene. The X-axis represents the log10 of the gene in the control group (the average ex-pression level), and the Y-axis represents the log10 of the gene in the experimental group (the average ex-pression level). The point of gray represents the gene with no significant difference, the point of orange rep-resents the gene with significant up-regulation compared with the control group, and the point of green repre-sents the gene with significant down-regulation compared with the control group. There are two screening thresholds for the screening criteria for differentially expressed genes: 1.0 or -1.0 when the expression multi-ple is taken as a logarithm. [Refer to the attachment for gene pathway information](https://static.igem.org/mediawiki/2019/6/6e/T--BM-AMU--Res2.zip)

![Experiment]( https://static.igem.org/mediawiki/2019/2/2e/T--BM-AMU--fig7.jpg)

**Figure 7: Transcriptome landscape among EMT cell subtypes.**

**Figure 7:** A total of 2943 genes were included in the different gene sets between samples. The expression of genes in the different gene sets between samples was standardized by Z score calculation method. The standardized expression profiles of differentially expressed gene sets were analyzed by -hierarchical clustering method, and the form of thermal map of clustering results was expanded. [Refer to the attachment for gene information](https://static.igem.org/mediawiki/2019/4/41/T--BM-AMU--Res3.csv)

![Experiment]( https://static.igem.org/mediawiki/2019/d/df/T--BM-AMU--fig8.jpg)

**Figure 8: Cluster Map of Differential Genes among EMT Cell Subtypes.**

**Figure 8:** Based on the results of figure 7, the Euclidean distance between genes was calculated by the standardized differential gene set expression, and the K-means cluster analysis was carried out. According to the graph, the differential gene sets between groups can be divided into four sets, and the genes of each set have simi-lar expression trends. In the future, we will enrich genes with similar trends in EMT process from known EMT-related genes, and conduct a physical investigation on them in order to achieve a more complete landscape of transcriptome changes in EMT process. [Refer to the attachment for gene information](https://static.igem.org/mediawiki/2019/3/3b/T--BM-AMU--Res4.csv)

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