Difference between revisions of "Part:BBa K2908676"

Revision as of 16:19, 21 October 2019

s(GATA3)p-GAD-miR101-BS

The CSU_CHINA iGEM team 2019 designed a fusion protein consisting of GAD and miR101-BS for sequence-specific expression of GAD.Therefore,we used our p(GATA3)p(BBa_K2908000) as a specific promoter. At the same time we used our miR101-BD(BBa_K2908668) to bind different concentrations of miR101 and fuse it to 3' end so that can regulate the transactivation domain of GAD.In this way we can ensure the specificity and independence of this protein.
New part: s(GATA3)p-GAD-miR101-BD (BBa_K2908676) [GAD is totally the same as Gal4-VP16]
Existing part: CMV-Gal4-VP16 (BBa_K2580666)
The existing part has low specificity in the TNBC cells compared to normal cells although the team members who created the part stated that it is a cancer-specific part.
As for this new part we created this year, it contains the brand new synthetic promoter we designed (BBa_K2908000),the existing sequence in the previous iGEM parts, GAD (Gal4-VP16), and the new designed part miR101-BD.
In our project, we wanted to make the expression of GAD to be specific in Triple negative breast cancer cells (TNBC cells). After communicating with the team creating the part who says that in breast cancer, this new part has higher efficiency in cancer cells than normal cells, we tested the efficiency of GAD expression by co-transfecting the CMV-Gal4-VP16 plasmid and pGL4.22-G8p (pGL4.22 has a luciferase gene after G8p, a promoter can be driven by Gal4-VP16) into TNBC cells (cancer cells) and normal cells. A in the figure below shows the results.
Then, we exchange the promoter CMV into s(GATA3)p, and added a miR101-BD in the 3’UTR of the gene GAD (Gal4-VP16), as miR101 is low expressed in the cancer cells while high in the normal cells [1] (the binding of miR101 to miR101-BD can inhibit the expression of GAD protein, then to down-regulate the efficiency of G8p). We used the same method to test the efficiency of our part, that is, co-transfecting the s(GATA3)p-GAD-miR101-BD plasmid and pGL4.22-G8p, and doing the luciferase assay [2]. The figure B shows the results. We also compared the specificity between CMV-Gal4-VP16 and s(GATA3)p-GAD-miR101-BD (C), which indicated that our part dramatically improved the function in specifically regulating the expression of GAD.
(A) The relative strength of previous part ‘CMV-Gal4-VP16’. (B) The relative strength of the new part ’s(GATA3)p-GAD-miR101-BD’. (C) The comparison of the specificity in cancer cells between new part and the previous part.
The part improved in the specificity in Triple negative cancer (TNBC).
Sequence and Features

References:
[1] H Guan et al.  Int J Mol Med (2016), MicroRNA-101 inhibits cell proliferation and induces apoptosisby targeting EYA1 in breast cancer.
[2] Solberg N1, Krauss S. , Methods Mol Biol. 2013;977:65-78. Luciferase assay to study the activity of a cloned promoter DNA fragment.