Team:BM-AMU- 2019.igem.org

Demonstrate

We have successfully achieved:

■ √ construction of Ecad-tdTomato monitor on WA09

■ √ construction and testing of controllers based on Tet-on/Tamoxifen-ERT systems

■ √ acquisition of specific EMT cells by controllers & monitor

■ √ acquisition of dynamic transcriptional map based on FACS-seq

■ √ designing of predicting tool based on Supporting Vector Machine (SVM)

And the followings are on the way:

■ construction of Ncad-eGFP monitor on WA09

■ optimization of controllers

■ optimization of predicting tool

1. Construction of monitor

In order to establish the fluorescence model, we need to transfer pBKS-hE-Cadherin-tdTomato and pBKS-hN-Cadherin-eGFP and supporting plasmid PX330 into WA09 cells to function. After verification, our hE-Cadherin-tdTomato cell model was constructed successfully, but the hN-Cadherin-eGFP model is on the way, data not shown. The following data are the main figures for in the verification process of the hE-Cadherin-tdTomato model.

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

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 (Figure 1A and 1B). 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（Figure1C）.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 (Figure1D). 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(Figure1E).The above results indicate that tdTomato inserts into the correct site and can function normally.

2. Construction of controller

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

Figure 2：Tamoxifen induced expression system works well.

Transfected plasmids 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 induced expression 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 induced expression system works well.

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 which has stable Tet-on system, lentivirus was used for transfection. LC-TetO-IN-hSnail1(BBa_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）). In order to obtain EMT cells in different states by changing DOX treatment，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 mesenchyme 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 strength comparison. To provide CAG and EF1a with quantitative data. After treatment（Details are described in the Experiment), the relative strengths of four promoters 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 4A（right）The results obtained by FACS are shown in the Figure 4A（left). The absolute value of fluorescence is median fluorescence intensity with an arbitrary unit.

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, 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. The results of the FACS illustrates that without induction with doxycycline, GFP in the original system is still expressed. However, comparing with rtTA and TetR 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 protein 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 captured and analyzed the differences of cell fluorescence phenotypes. Therefore, we are interested in the difference of fluorescence phenotype induced by SNAIL1 overexpression compared with the difference of transcriptome 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 sequenced 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 significance 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 expression level), and the Y-axis represents the log10 of the gene in the experimental group (the average expression level). The point of gray represents the gene with no significant difference, the point of orange represents the gene with significant up-regulation compared with the control group, and the point of green represents 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 multiple 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 similar 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/Demonstrate