Part:BBa_K2591014

TCF-TATA-GFP

TCF-TATA-GFP

Usage and Biology

The WNT signaling pathway is one of the cellular signal transduction pathways, which begins with reception of WNT ligands in plasma membrane, and initiate a series of downstream events. If there is no reception of WNT ligands, the subunits of disruption complex is free, and it can bind the downstream protein β-catenin in cytoplasm, mediating its degradation, which means there is no more downstream events.(Fig.1)

Figure1. Condition of no WNT signal

However, when WNT ligands bind to the receptor Frizzled, the disruption complex is fixed by WNT-Frizzled in plasma membrane, which leads to release and accumulation of β-catenin. Then, the β-catenin can finally translocated into the nucleus and acts as transcriptional co-activator of transcriptional factor TCF. The β-catenin-TCF complex then interacts with TCF transcriptional response element(TCF TRE) and start the transcription of WNT target genes.(Fig.2)

Figure2. Condition of WNT on

In the absence of β-catenin, TCF acts as a repressor of WNT target genes by occupying the TCF TRE, but β-catenin can convert the TCF to a transcriptional activator of the same genes. This novel process can be extract from nature and make up a reporter system when we replace the target genes into a fluorescence gene like GFP. When this whole construct stably insert to a receptor cell line, it can sense WNT signal and give green fluorescence.(Fig.3)

Figure3. GFP followed by TCF TRE

The part we construct is improved from GFP part in Registry(link to GFP part https://parts.igem.org/Part:BBa_E0040), in front of GFP, we add two TCF transcriptional response elements and a TATA box.(Fig.4)

Figure4. The construct of TCF-TATA-GFP in pSB1C3

Characterization

We first designed primers and construct the plasmid in Snapgene, then according to the protocols which we write previously, we did some molecular cloning experiments. Finally we got the plasmid and sent to Sanger sequencing. Forward(VF2) and reverse(VR) sequencing results show that we have successfully got the right plasmid.(Fig.4)

Figure4. Forward and reverse sequencing results aligned with designed plasmid After verification in sequence level, we designed experiments to verify its function. We have stably integrated this construct into the genome of our reporter cell line by lentiviral infection. At the same time, we have used the original part(BBa_E0040) to do the same experiments, the fluorescence images are shown in Fig5. And for TCF-GFP cells, we added Wnt3a to the culture medium, after several hours, we can observe green fluorescence under microscope(Fig.6) and also, we can see a shift in fluorescence activated cell sorting(FACS) experiments.(Fig.7)

Figure5. Fluorescence image of original GFP part(BBa_E0040 https://parts.igem.org/Part:BBa_E0040)

Figure6. Fluorescence image of WNT activation by Chir99021(GSK3β inhibitor) and Wnt3a

Figure7. FACS results show GFP intensity shift after adding Wnt3a

Application

This TCF-TATA-GFP construct can be used to indicate the activation of WNT pathway, which largely related carcinogenesis. Therefore, it’s a powerful indicator in cancer pathway researches.(Akiri, et al, 2009) Besides this, it can indicate the extracellular Wnt3a protein, acting as perfect reporter system. This method of transcriptional response element(TRE) coupled with a fluorescence gene gives a new window for biosensor in synthetic biology. Especially when the chassis organisms are eukaryotic cells. It may inspire more iGEMers to design project based on eukaryotic cells.

References

Akiri, G., Cherian, M. M., Vijayakumar, S., Liu, G., Bafico, A., & Aaronson, S. A. (2009). Wnt pathway aberrations including autocrine Wnt activation occur at high frequency in human non-small-cell lung carcinoma. Oncogene, 28(21), 2163.

Blauwkamp, T. A., Nigam, S., Ardehali, R., Weissman, I. L., & Nusse, R. (2012). Endogenous Wnt signalling in human embryonic stem cells generates an equilibrium of distinct lineage-specified progenitors. Nature communications, 3, 1070.

LEF, G. W., & GFP, T. D. (2002). LEF/TCF-Dependent, Fluorescence-Based Re-porter Gene Assay For Wnt Signaling. BioTechniques, , 32, 1000-1002