In order to reduce gene expression leakage for more efficient regulation, we have constructed a "AND gate" system regulated by lactose and estradiol. In the experiment, the introduction of the EBD protein significantly reduced the leakage expression of the lactose regulation system [1], and we chose to use sfGFP to characterize the expression of this protein. This estradiol regulation system will be uploaded to the iGEM Registry for the first time, providing new regulatory ideas for other iGEM teams.

EBD stands for estrogen-binding domain. We connect sfGFP with EBD through a linker to express the fusion protein. In the presence of estradiol, estradiol binds to EBD, activating the fusion protein, which can correctly fold, and at this time sfGFP emits a fluorescent signal. When there is no estradiol signal, the sfGFP-EBD fusion protein unfolds, binds to Hsp90, and thus cannot correctly fold, and the fluorescent protein has no signal.

At the same time, between the EBD and the promoter, the gene of a specific protein can also be inserted, connecting it with sfGFP, EBD protein through a linker, and they will exist in the form of a fusion protein. In the presence of estradiol, estradiol binds to EBD, activating the fusion protein, which can correctly fold, and at this time sfGFP emits a fluorescent signal, and the specific protein will also correctly express its function. When there is no estradiol signal, the sfGFP-EBD fusion protein unfolds, binds to Hsp90, and thus cannot correctly fold, and the fluorescent protein has no signal, and the specific protein will also fail to function.

[1] Ma, Y., Su, S., Fu, Z. et al. Convenient synthesis and delivery of a megabase-scale designer accessory chromosome empower biosynthetic capacity. Cell Res 34, 309–322 (2024).