Part:BBa_K2908000

Sructure

- We identified genes that were differentially expressed in human triple negative breast cancer (TNBC) cells compared to healthy tissues from the publicly available The Cancer Genome Atlas (TCGA) database [1] using R (version 3.0.2). We followed standard procedures to calculate differentially expressed genes between cancer cases compared to controls using t test. The p values were corrected for multiple testing using the Bonferroni correction. We then selected genes that were previously identified as transcription factors (TFs) from the literature. From these genes (ESR1, FOXA1, GATA3, E2F1, PTTG1, UHRF1, FOXM1, MYBL2, PITX1), we selected the ones that verified as the most specific in MDA-MB-231 cells (TNBC cell line) compared to HBL-100 cells (normal breast cell line). (Refer to the Project/Demonstrate page in our wiki to see the results. Address:https://2019.igem.org/Team:CSU_CHINA/Demonstrate)
- We then determined the binding motifs for ovarian cancer-enriched TFs using GREAT, MEME, JASPAR and MOTIFMAP [2-5] and assembled them into our synthetic promoters.
- Construction: We use the JASPER to get the TF binding sites (TF-BSs), and add different spacers between the tandem repeats of TF-BSs to avoid creating new binding sites in order to ensure the specificity.

This figure shows the results we browse using JASPAR by input homo sapiens GATA3.

Then we add a strong and short promoter named lateADEp to ensure the strength of the whole promoter.
- References:
[1] Barretina, J., Caponigro, G., Stransky, N., Venkatesan, K., Margolin, A.A., Kim, S., Wilson, C.J., Leha´ r, J., Kryukov, G.V., Sonkin, D., et al. (2012). The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483, 603–607.
[2] Bailey, T.L., Boden, M., Buske, F.A., Frith, M., Grant, C.E., Clementi, L., Ren, J., Li, W.W., and Noble, W.S. (2009). MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 37, W202–W208.
[3] Bell, D., Berchuck, A., Birrer, M., Chien, J., Cramer, D.W., Dao, F., Dhir, R., DiSaia, P., Gabra, H., Glenn, P., et al.; Cancer Genome Atlas Research Network (2011). Integrated genomic analyses of ovarian carcinoma. Nature 474, 609–615.
[4] McLean, C.Y., Bristor, D., Hiller, M., Clarke, S.L., Schaar, B.T., Lowe, C.B., Wenger, A.M., and Bejerano, G. (2010). GREAT improves functional interpretation of cis-regulatory regions. Nat. Biotechnol. 28, 495–501.
[5] Sandelin, A., Alkema, W., Engstro¨ m, P., Wasserman, W.W., and Lenhard, B. (2004). JASPAR: an open-access database for eukaryotic transcription factor binding profiles. Nucleic Acids Res. 32, D91–D94.