Team:Slovenia/TheSwitchControls

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Controls

Inducible TAL regulators

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We improved the induction system we previously used 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.

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.

Specificity of TAL effectors

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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.

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).

Test of minimal promoter leakage amplified by autoactivator

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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.

[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)

Test of bistability of the positive feedback loop

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To determine whether our improved switch is bistable, we transfected HEK293T cells with all four constructs comprising the bistable switch with a positive feedback loop, without plasmids comprising the induction system. We observed fluorescence of both reporters as cells randomly shift to one of the two possible states [A]. We could observe that each reporter was expressed in different cells as shown in the overlay and no fluorescence colocalization was determined [B]. This clearly demonstrates bistability of the positive feedback loop in contrast to the simple mutual repressor switch which demonstrated high degree of fluorescence overlap (Figure 7 of the Mutual repressor section).

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

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 and state II

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In order to remotely control the switch by small moleclar weight inducers, 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 (second 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).

[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)

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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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Next we tested the response of the switch when all of the first level plasmids, but second level plasmids (E:KRAB and PIP:KRAB) 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 inducer-dependant 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).

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)

The switch coupled with both induction systems - NOT INDUCED

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To finally prove the bistability of the complete switch (including the complete induction systems) we transfected the cell with all of the plasmids and left them unstimulated. With microscopy and flow cytometry we observed two populations of cells both expressing only one of the fluorescent reporters, confirming our expectations of bistability as even in the absence of inducers the system stochastically switches to one or the other of the two clearly defined stable states.

[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, PCMV_[TRE]_TALA:KRAB, PCMV_[TRE]_TALB:VP16 (all 40ng) PCMV_PIP:KRAB, PCMV_E:KRAB (both 80 ng).

The switch coupled with both induction systems - both inducers present simultaneously

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Some of the judges at the European Jamboree were curious as to what would happen if the system is stimulated with both inducers simultaneously. A combination of both signals in electronic circuits results in an undefined state. Experimental results for our switch suggest that both reporters are effectively repressed, as we determined that a transcription repressor with the KRAB domain is able to overpower the effect of a transcription activator with the VP16 domain.

[A]_PMIN_TALB:KRAB (10 ng), [A]_PMIN_TALA:VP16_t2A_BFP (2 ng), [B]_PMIN_TALA:KRAB_t2A_mCitrine (10 ng), [B]_PMIN_TALB:VP16, (2 ng), PCMV_[PIR]_TALB:KRAB (20 ng), PCMV_[PIR]_TALA:VP16 (5 ng), PCMV_[ETR]_TALA:KRAB (20 ng), PCMV_[ETR]_TALB:VP16, ( 5 ng), PCMV_PIP:KRAB, PCMV_E:KRAB (both 200 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_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16, PCMV_[TRE]_TALA:KRAB, PCMV_[TRE]_TALB:VP16 (all 40ng) PCMV_PIP:KRAB, PCMV_E:KRAB (both 80 ng).

Stimulation

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We prepared a switch with inducible systems comprising CL1-PEST degradation tags (see above), 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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