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Improved Part

Composite Part

Section	Name	Type	Description
New part	BBa_K2908676	DNA	s(GATA3)p-GAD-miR101-BS
Existing part	BBa_K2580666	DNA	CMV-Gal4-VP16

New part

s(GATA3)p-GAD-miR101-BD (BBa_K2908676) [GAD is totally the same as Gal4-VP16]

The part improved in the specificity in Triple negative cancer (TNBC).

Existing part

CMV-Gal4-VP16 (BBa_K2580666)

The 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 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.

Appendix: Brief introduction of luciferase assay

The Luciferase Assay System is substantially improved over conventional assay methods in both sensitivity and simplicity. Light is produced by converting the chemical energy of luciferin oxidation through an electron transition, forming the product molecule oxyluciferin. Firefly luciferase, a monomeric 61kDa protein, catalyzes luciferin oxidation using ATP-Mg2+ as a cosubstrate. In the conventional assay for luciferase, a flash of light is generated that decays rapidly after the enzyme and substrates are combined. The Luciferase Assay System incorporates coenzyme A (CoA) for improved kinetics, allowing greater enzymatic turnover and resulting in increased light intensity that is nearly constant for at least 1 minute. The Luciferase Assay System yields linear results over at least eight orders of magnitude. Less than 10-20 moles of luciferase have been detected under optimal conditions. Generally, 100-fold greater sensitivity can be achieved over the chloramphenicol acetyltransferase (CAT) assay.

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.