Team:Slovenia/TheSwitchControls

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Controls

Inducible TAL regulators.

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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 improved the induction system we previously used (link mutual repressor) by placing the binding sites upstream of the promoter and by fusing CL1-PEST degradation tags to TAL effectors. 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 strong repression of the reporter upon induction, confirming that the inducible TAL repressor is expressed and functions as expected.

Specificity of TAL effectors.

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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.

Test of minimal promoter leakage amplified by autoactivator.

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[A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP (both 20 ng)	[B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 20 ng)

In order to investigate the leakage of minimal promoters in the switch, we performed two controls that included the plasmids of one or the other state of the switch but no induction system. We observed flourescence corresponding to the transfected plasmids in few cells, indicating some promoter leakage. Even though we determined a minimal leakage of the minimal promoter this minimal promoter leakage can nevertheless initiate transcription of the activator which in turn triggers the positive feedback loop and causes further activation of the system and expression of the reporter. Leakage of the minimal promoter and amplification of gene expression were detected for both states of the switch which also indicates that there is no difference if the fluorescent reporter protein is linked to an activator or to a repressor.

Test of bistability of the positive feedback loop.

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[A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, [B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 20 ng)

In order to investigate the leakage of minimal promoters in the switch, we performed two controls that included the plasmids of one or the other state of the switch but no induction system. We observed flourescence corresponding to the transfected plasmids in few cells, indicating some promoter leakage. Even though we determined a minimal leakage of the minimal promoter this minimal promoter leakage can nevertheless initiate transcription of the activator which in turn triggers the positive feedback loop and causes further activation of the system and expression of the reporter. Leakage of the minimal promoter and amplification of gene expression were detected for both states of the switch which also indicates that there is no difference if the fluorescent reporter protein is linked to an activator or to a repressor.

Illustration of the readout of results of the flow cytometry with transiently transfected mammalian cells that are able to produce two fluorescent reporters.

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Schematic representation of populations of cells transfected with the switch as measured by flow cytometry. Upper left quadrant (Q1): cells producing only BFP; lower right quadrant (Q3): cells producing only mCitrine; right top quadrant (Q2): cells producing both BFP and mCitrine; lower left quadrant (Q4): nontransfected cells and cells that produce neither BFP nor mCitrine. The efficiency of mammalian cell transfection was typicaly between 30-50%.

Nontransfected cells.

Induction of state I.

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[A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, [B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 5 ng), PCMV_[ETR]_TALA:KRAB, PCMV_[ETR]_TALB:VP16, (all 10 ng)

A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, [B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 20 ng), PCMV_[TRE]_TALA:KRAB, PCMV_[TRE]_TALB:VP16, (all 40ng)

In order to remotely control the switch by small moleclar weight inductors, we added an induction system, comprised of two operons that express the appropriate TAL repressor and activator from an inducible promoter (first level plasmids), and one operon that constitutively expresses an inducer-dependent transcription factor (zero-level plasmid). In the absence of this transcription factor, the inducible promoters act as constitutive promoters. In order to enable switching between two distinct states, two parallel induction systems are required (comprising six operons). We tested the response of the switch when only the first level plasmids of one of the induction systems are added. As shown by fluorescence measurements, the switch shifted into the expected state; plasmids of the erithromycine or tetracycline system shift the switch into state I (mCitrine expression) while plasmids of the pristinamycine system shift the switch into state II (BFP expression).

Induction of state II.

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[A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, [B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16 (all 5 ng), PCMV_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16 (both 10ng)

A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, [B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 20 ng), PCMV_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16, (all 40ng)

The switch coupled with both induction systems.

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PCMV_PIP:KRAB (200ng), [A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, [B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 5 ng), PCMV_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16, PCMV_[ETR]_TALA:KRAB, PCMV_[ETR]_TALB:VP16, (all 10 ng),

PCMV_E:KRAB (200 ng)

Next we tested the response of the switch when all of the first level plasmids, but plasmids of only one of the induction systems are present. This should result in the constitutively active promoters on the first level plasmids without their corresponding repressors, and (in the absence of inducer) constitutively repressed promoters on the first level plasmids of the complete induction system. A missing repressor thus in effect simulates a constitutive presence of the corresponding inducer. As expected, the system behaves in the same way as when it is induced. Without the addition of PIP:KRAB, the system expresses BFP (state I) and without the addition of E:KRAB the system expresses mCitrine (state II).

Stimulation

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We prepared a switch with inducible systems comprising CL1-PEST degradation tags (see figure/control 1), which should allow faster switching between states. We tested how these improved induction systems induce one state of the switch or the other. We also tested if the switch retains the stable state if the inducer is removed.

Cells constantly stimulated with erythromycin (day 5 after the transfection). HEK293T cells were cotransfected with the following plasmids: [A]_PMIN_TALB:KRAB (21 ng), [A]_PMIN_TALA:VP16_t2A_BFP (7 ng), [B]_PMIN_TALA:KRAB_t2A_mCitrine (21 ng), [B]_PMIN_TALB:VP16 (7 ng), [PIR]_PCMV_ CL1-PEST:TALB:KRAB (30 ng), [PIR]_PCMV_ CL1-PEST:TALA:VP16 (10 ng), [ETR]_PCMV_ CL1-PEST:TALA:KRAB (30 ng), [ETR]_PCMV_ CL1-PEST:TALB:VP16 (10 ng), PCMV_PIP:KRAB, PCMV_E:KRAB (both 300 ng). Erythromycin was added to final concentration of 2 µg/ml. Fluorescence was measured by flow cytometry 5 days after transfection.

Cells constantly stimulated with pristinamycin (day 5 after the transfection). HEK293T cells were cotransfected with the following plasmids: [A]_PMIN_TALB:KRAB (21 ng), [A]_PMIN_TALA:VP16_t2A_BFP (7 ng), [B]_PMIN_TALA:KRAB_t2A_mCitrine (21 ng), [B]_PMIN_TALB:VP16 (7 ng), [PIR]_PCMV_ CL1-PEST:TALB:KRAB (30 ng), [PIR]_PCMV_ CL1-PEST:TALA:VP16 (10 ng), [ETR]_PCMV_ CL1-PEST:TALA:KRAB (30 ng), [ETR]_PCMV_ CL1-PEST:TALB:VP16 (10 ng), PCMV_PIP:KRAB, PCMV_E:KRAB (both 300 ng). Pristinamycin was added to final concentration of 2 µg/ml. Fluorescence was measured by flow cytometry 5 days after transfection.

Inducer removal

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After removal of erythromycin the switch stably expresses mCitrine HEK293T cells were cotransfected with the following plasmids: [A]_PMIN_TALB:KRAB (21 ng), [A]_PMIN_TALA:VP16_t2A_BFP (7 ng), [B]_PMIN_TALA:KRAB_t2A_mCitrine (21 ng), [B]_PMIN_TALB:VP16 (7 ng), [PIR]_PCMV_ CL1-PEST:TALB:KRAB (30 ng), [PIR]_PCMV_ CL1-PEST:TALA:VP16 (10 ng), [ETR]_PCMV_ CL1-PEST:TALA:KRAB (30 ng), [ETR]_PCMV_ CL1-PEST:TALB:VP16 (10 ng), PCMV_PIP:KRAB, PCMV_E:KRAB (both 300 ng). Erythromycin was added to final concentration of 2 µg/ml. After one day the cell medium was changed, removing the erythromycin. Fluorescence was measured by flow cytometry 5 days after transfection.

After removal of pristinamycin the switch stably expresses mCitrine HEK293T cells were cotransfected with the following plasmids: [A]_PMIN_TALB:KRAB (21 ng), [A]_PMIN_TALA:VP16_t2A_BFP (7 ng), [B]_PMIN_TALA:KRAB_t2A_mCitrine (21 ng), [B]_PMIN_TALB:VP16 (7 ng), [PIR]_PCMV_ CL1-PEST:TALB:KRAB (30 ng), [PIR]_PCMV_ CL1-PEST:TALA:VP16 (10 ng), [ETR]_PCMV_ CL1-PEST:TALA:KRAB (30 ng), [ETR]_PCMV_ CL1-PEST:TALB:VP16 (10 ng), PCMV_PIP:KRAB, PCMV_E:KRAB (both 300 ng). Pristinamycin was added to final concentration of 2 µg/ml. After one day the cell medium was changed, removing the pristinamycin. Fluorescence was measured by flow cytometry 5 days after transfection.

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