Team:BM-AMU- 2019.igem.org

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.

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.

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.

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

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.

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.

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

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

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

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

Team:BM-AMU/Results