Team:Slovenia/TheSwitchControls

Controls

Inducible TAL regulators.

Erythromycin induces the expression of a TAL repressor, which represses a luciferase reporter. HEK293T cells were cotransfected with transfection control (10 ng Renilla luciferase), [AB]_PCMV_fLuc reporter and ETR_PCMV_PEST-CL1-TALA:KRAB (both 10 ng) and E:KRAB (100 ng). Two hours post-transfection the cells were stimulated with 2 µg/ml erythromycin.	Pristinamycin induces the expression of a TAL repressor, which represses a luciferase reporter. HEK293T cells were cotransfected with transfection control (10 ng Renilla luciferase), [AB]_PCMV_fLuc reporter and PIR_PCMV_PEST-CL1-TALA:KRAB (borh 10 ng) and PIP:KRAB (100 ng). Two hours post-transfection the cells were stimulated with 2 µg/ml pristinamycin.

We adapted the original induction systems for erythromycin and pristinamycin which were kindly provided by prof. Martin Fussenegger. Before using them in our TAL-based switches we had to determine their functionality. To test them we cotransfected inducible repressors with a reporter and observed the repression with and without the addition of the appropriate inducer. The tests show string repression of the reporter upon induction, confirming that the inducible TAL repressor is expressed and functions as expected.

Specificity of TAL effectors.

TAL activator specifically recognizes its corresponding DNA-binding site. HEK293T cells were cotransfected with transfection control PCMV_ mCherry (10 ng), PCMV_TALA:VP16 [a,b] or PCMV_TALB:VP16 (c) and reporter [AB]_PCMV_mCit (a), [CB]_PCMV_mCit (b), or [AC]_PCMV_mCit (all 10 ng) (b). Fluorescence was detected only in cells cotransfected with the specific TAL effector and its corresponding DNA-binding site (a), while cross-reactivity with multiple copies of other binding sites was not observed (b and c).

For the switch to function properly, all of TAL effectors need to exhibit high specificity and orthogonality. We tested different combinations of TAL operons and TAL activators (Figure) to control for any cross-reactivity and binding specificity. When a reporter under the TAL operon was cotransfected with its matching TAL activator, fluorescence was detected. When the transfected reported plasmid‘s operon did not match the cotransfected TAL activator, no flourescence was detected even if it contained 10 copies of two other TAL binding sites. This demonstrates that TAL regulators bind and exert activity specifically at their binding sites and high degree of orthogonality.