A precisely adjustable, variation-suppressed eukaryotic transcriptional controller to enable genetic discovery
- Computational Systems Biology and Swiss Institute of Bioinformatics, ETH Zurich, Switzerland
- Division of Basic Sciences, Fred Hutchinson Cancer Research Center, United States
Figures
Figure 1 with 1 supplement
Repression of engineered PTDH3 derivatives by TetR.
(A) Structure of the starting promoter, PTDH3. Diagram shows the nucleotide positions of the binding sites for the endogenous transcription factors Rap1 and Gcr1, the TATA-sequence, and the …
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Figure 1—source data 1
Numerical data for Figure 1.
- https://cdn.elifesciences.org/articles/69549/elife-69549-fig1-data1-v2.zip
Figure 1—figure supplement 1
A promoter with three tetO1 sequences in the UAS of PTDH3 is only minimally repressed by TetR.
We constructed P3tet, a PTDH3 derivative that carried three tetO1 TetR binding sites, adjacent to the endogenous transcription binding sites (one for Rap1, two for Gcr1) in the UAS. We used this to …
Figure 2 with 4 supplements
Comparison of the three controller architectures.
(A) Genetic elements of the different controller architectures used in these experiments. The * next to TetR indicates SV40 Nuclear Localization Sequence and flat headed arrows indicate repression. …
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Figure 2—source data 1
Numerical data for Figure 2 panels B and D.
- https://cdn.elifesciences.org/articles/69549/elife-69549-fig2-data1-v2.zip
Figure 2—figure supplement 1
Variation in expression for the SR, AR, and cAR architectures.
We calculated the CoV in fluorescence signal using the data shown in main Figure 2B, to test whether the volume-corrected RSD measure described in Appendix 2 and Figure 2—figure supplement 2 agrees …
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Figure 2—figure supplement 1—source data 1
Numerical data for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/69549/elife-69549-fig2-figsupp1-data1-v2.zip
Figure 2—figure supplement 2
Single-reporter VIV measure of CCV in expression.
Scatter plot of flow cytometry cell volume proxy vs. Citrine fluorescence in a strain where Citrine is constitutively expressed from PTDH3 at the LEU2 locus (Y2683) shows a positive correlation …
Figure 2—figure supplement 3
Autorepression loop reduces cell-to-cell variability at intermediate concentrations of aTc.
Plot of flow cytometry cell volume proxy vs Citrine fluorescence in whole (ungated) populations of cells of SR, AR, and cAR architectures (Y2663,2674,2741) grown for 7 hr at different aTc …
Figure 2—figure supplement 4
Single-reporter VIV measure of variation in expression from native yeast promoters.
We measured Citrine expression and its VIV in otherwise isogenic haploid cells that carried integrated constructs at various auxotrophic marker loci, in which the indicated native yeast promoters …
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Figure 2—figure supplement 4—source data 1
Numerical data for Figure 2—figure supplement 4.
- https://cdn.elifesciences.org/articles/69549/elife-69549-fig2-figsupp4-data1-v2.zip
Figure 3 with 3 supplements
Repressor optimization to abolish P7tet.1 basal expression.
A) Testing repression by the TetR-Tup1 fusion. The top diagram indicates the genetic elements of the SR architecture used to test the ability of the TetR-Tup1 fusion to abolish basal expression from …
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Figure 3—source data 1
Numerical data for Figure 3.
- https://cdn.elifesciences.org/articles/69549/elife-69549-fig3-data1-v2.zip
Figure 3—figure supplement 1
The zeroing repressor TetR-nls-Tup1 does not affect the induction speed of P7tet.1.
Citrine signal after induction in P7tet.1-Citrine strains. Both strains carried a P7tet.1-Citrine integrated at the LEU2 locus, in conjunction with two different repressors. One strain (red, Y2674) …
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Figure 3—figure supplement 1—source data 1
Numerical data for Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/69549/elife-69549-fig3-figsupp1-data1-v2.zip
Figure 3—figure supplement 2
A TetR-nls-Tup1 fusion protein fully represses expression when binding only at the UAS, but not only at the TATA.
Strains carried a PACT1 construct that directed the expression of TetR-nls-Tup1 integrated at HIS3 locus. Y2702 (blue line) also carried P3tet-Citrine construct, which bears three TetR binding sites …
Figure 3—figure supplement 3
Low level TetR-nls-Tup1 expression results in incomplete repression of P7tet.1.
Fluorescence signal from a strain where a P7tet.1-Citrine construct was integrated at the LEU2 locus and repressed by PREV1-driven TetR-nls-Tup1 integrated at HIS3 and P7tet.1 driven TetR integrated …
Figure 4 with 9 supplements
Controlled gene expression from WTC846.
(A) Architecture of WTC846. The final WTC846 system is composed of a single integrative plasmid bearing TetR and TetR-Tup1 driven by the promoters indicated. This plasmid was integrated at the URA3 …
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Figure 4—source data 1
Numerical data for Figure 4 panels B and D.
- https://cdn.elifesciences.org/articles/69549/elife-69549-fig4-data1-v2.zip
Figure 4—figure supplement 1
Direct observation of dose response for WTC846-controlled protein expression.
The strain WTC846::citrine (Y2759), in which both TetR and Citrine were expressed from P7tet.1, and TetR-nls-Tup1 was expressed constitutively from PRNR2, was grown in YPD to stationary phase with …
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Figure 4—figure supplement 1—source data 1
Raw and uncropped images for western blots in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/69549/elife-69549-fig4-figsupp1-data1-v2.zip
Figure 4—figure supplement 2
Dose response of WTC846-controlled expression in cells grown in different media.
(A) Strain carrying WTC846::citrine construct (Y2759) grown in different media. Dots represent the median fluorescence of each population, and the lines were fitted using a five parameter …
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Figure 4—figure supplement 2—source data 1
Numerical data for Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/69549/elife-69549-fig4-figsupp2-data1-v2.zip
Figure 4—figure supplement 3
WTC846-directed expression does not affect gross measures of cell physiology.
We measured the effect of WTC846 on growth rate and on cell viability by ability of single cells to form colonies. To do so, we used the WTC846:: strain Y2761, in which a construct bearing P7tet.1-Te…
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Figure 4—figure supplement 3—source data 1
Raw data and raw plate images for Figure 4—figure supplement 3.
- https://cdn.elifesciences.org/articles/69549/elife-69549-fig4-figsupp3-data1-v2.zip
Figure 4—figure supplement 4
Cell-to-cell variation of WTC846-driven expression during induction.
We used data from the Time Dependent Dose Response (TDDR) experiment shown in Figure 4B to calculate single reporter VIV during the course of induction of the WTC846::citrine strain (Y2759) at …
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Figure 4—figure supplement 4—source data 1
Numerical data for Figure 4—figure supplement 4.
- https://cdn.elifesciences.org/articles/69549/elife-69549-fig4-figsupp4-data1-v2.zip
Figure 4—figure supplement 5
Peak CCV in SR strains corresponds to higher doses at higher expression levels of TetR-nls-Tup1.
We used data from the dose response experiment shown in Figure 3C to calculate single-reporter VIV to quantify CCV as described in the Appendix 2. We induced three strains (Y2669, Y2676, and Y2717) …
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Figure 4—figure supplement 5—source data 1
Numerical data for Figure 4—figure supplement 5.
- https://cdn.elifesciences.org/articles/69549/elife-69549-fig4-figsupp5-data1-v2.zip
Figure 4—figure supplement 6
Fluorescence and volume of the WTC846::citrine strain induced with different aTc concentrations.
We grew cells of WTC846::citrine strain (Y2759) to exponential phase, and measured Citrine fluorescence with flow cytometry 7 hr after induction with aTc. The complete dose response is presented in …
Figure 4—figure supplement 7
CCV of WTC846-controlled expression in cells grown in different media.
CCV of WTC846 was measured in different media conditions using the WTC846::citrine strain (Y2759) and the VIV measure. Cells were grown in different media at different aTc concentrations, and …
Figure 4—figure supplement 8
Shutoff of WTC846 expression.
(A) Shutoff upon aTc removal from an exponentially growing, recently induced culture of WTC846::citrine (Y2759). Induction of the main culture (gray) with 600 ng/mL aTc was performed at time 0 and …
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Figure 4—figure supplement 8—source data 1
Numerical data for Figure 4—figure supplement 8.
- https://cdn.elifesciences.org/articles/69549/elife-69549-fig4-figsupp8-data1-v2.zip
Figure 4—figure supplement 9
Comparison of cell-to-cell variation between WTC846, a previously published, β-estradiol induced transcriptional control system, and expression driven by PGAL1.
Overlaid fluorescence and cell-to-cell variation curves are shown for each system (A) Dose response and cell-to-cell variation curves of the β-estradiol-induced LexA-hER-B112-control system after 24 …
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Figure 4—figure supplement 9—source data 1
Numerical data for Figure 4—figure supplement 9.
- https://cdn.elifesciences.org/articles/69549/elife-69549-fig4-figsupp9-data1-v2.zip
Figure 5 with 7 supplements
Controlled protein dosage of WTC846-driven yeast genes.
(A) The WTC846 architecture used, as in Figure 4A. Figure also shows the three extended Kozak sequences used to control translation efficiency. (B) WTC846 alleles of essential yeast genes show null …
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Figure 5—source data 1
- https://cdn.elifesciences.org/articles/69549/elife-69549-fig5-data1-v2.zip
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Figure 5—source data 2
Numerical data for Figure 5D.
- https://cdn.elifesciences.org/articles/69549/elife-69549-fig5-data2-v2.zip
Figure 5—figure supplement 1
Regulated protein dosage from WTC846 alleles controls growth on different solid media.
Cells of MATa haploid strains bearing genes whose expression was controlled by WTC846 were spotted onto solid media. Names of the genes are given on the left, which correspond to the ‘Name’ column …
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Figure 5—figure supplement 1—source data 1
Raw plate images for Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/69549/elife-69549-fig5-figsupp1-data1-v2.zip
Figure 5—figure supplement 2
WTC846-driven overexpression of Ipl1 prolongs G2/M and produces cells with >2 n ploidy.
We grew WTC846-K2::IPL1 (Y2789) and an otherwise isogenic parent strain (Y2769), where Ipl1 was under the control of its native promoter, in YPD with 400 ng/mL aTc for 18 hr. At each time point …
Figure 5—figure supplement 3
Adjustable protein dosage from WTC846 alleles of essential and metabolic genes controls growth rates in different liquid media.
Plots show growth curves of (A) Y2828/ WTC846-K3::PMA1 in SD Full, pH 4.5, (B) Y2773/WTC846-K3::TOR2 in YPD medium (in triplicate), (C) Y2849/WTC846-K3::TPI1 in YPD, and (D) Y2772/WTC846-K1::TOR2 …
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Figure 5—figure supplement 3—source data 1
Raw data for Figure 5—figure supplement 3.
- https://cdn.elifesciences.org/articles/69549/elife-69549-fig5-figsupp3-data1-v2.zip
Figure 5—figure supplement 4
Regulated clamped hypomorphic expression of WTC846::PMA1 allele causes cell separation defect.
We grew Y2828/WTC846-K3::PMA1 in SD Full media with the aTc concentrations indicated as ng/mL in the grey boxes above the panels. We fixed the samples with 70% ethanol for Sytox staining. We used …
Figure 5—figure supplement 5
Whi5 titration leads to increased cell volume without an increase in cell-to-cell variation.
(A) Expression of Whi5 was clamped at different levels by growth of Y2791, a haploid WTC846-K1::WHI5 strain, of Y2929, a WTC846-K1::WHI5/WTC846-K1::WHI5 diploid strain, and otherwise-isogenic …
Figure 5—figure supplement 6
Cell cycle arrest in the WTC846-K3::CDC20 strain.
Cells growing in 3 ng/mL aTc were arrested by aTc withdrawal to compare the time to reach arrest to that in Main Figure 5E, in which cells were grown in 20 ng/mL aTc and time to arrest was around 8 …
Figure 5—figure supplement 7
PGAL1-driven expression from a centromeric plasmid results in high cell-to-cell variation.
PGAL1 was used to drive Citrine expression in strain Y3281 from a centromeric plasmid. The strain was grown in synthetic media lacking uracil with 2% Raffinose and 2% Galactose. For comparison of …
Appendix 1—figure 1
Optimization of tetO1 placements and endogenous transcription factor binding sites to increase maximum activity of a TetR-repressible derivative of PTDH3.
(A) Diagram of PTDH3 shows the nucleotide positions of the binding sites for the endogenous transcription factors Rap1 and Gcr1, the TATA-sequence, and the transcription start site relative to the …
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Appendix 1—figure 1—source data 1
Numerical data for Appendix 1—figure 1.
- https://cdn.elifesciences.org/articles/69549/elife-69549-app1-fig1-data1-v2.zip
Appendix 2—figure 1
Sub-populations collected by FACS for validating the cell volume proxy measure.
Strain Y2683 was grown to exponential phase in YPD and was run through the sorter at a concentration of 2 million cells per mL. 10 separate gates were set on the FSC-W and SSC-H signals for …
Appendix 2—figure 2
Comparison of the flow-cytometry-based cell volume proxy to cell volume measured by Coulter counter.
10 sub-populations were collected from an exponentially growing culture used as constitutive Citrine expression control in other experiments (Y2683), using FACS as described in Figure Appendix …
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Appendix 2—figure 2—source data 1
Numerical data for Appendix 2—figure 2C.
- https://cdn.elifesciences.org/articles/69549/elife-69549-app2-fig2-data1-v2.zip
Appendix 3—figure 1
Effect of increased nuclear concentration and size of TetR on repression.
The top diagram indicates the genetic elements of the SR architecture used to test the ability of various TetR derivatives to abolish basal activity of P7tet.1. Diagrams to the left of the plots …
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Appendix 3—figure 1—source data 1
Numerical data for Appendix 3—figure 1.
- https://cdn.elifesciences.org/articles/69549/elife-69549-app3-fig1-data1-v2.zip
Appendix 4—figure 1
Simulations showing the relationship between production and degradation rates and time to steady state.
The ODE model presented in Appendix 4 was simulated with (A) constant production rate a = 0.2, and a varying degradation rate d, or (B) with constant degradation rate d = 0.005 and a varying …
Appendix 5—figure 1
The configuration of and an example of gene expression control by WTC846.
(A) Genetic elements of the WTC846 controller. On the integrative plasmid, TetR is driven by the P7tet.1, TetR-nls-Tup1 is driven by the RNR2 promoter. The promoter of the gene of interest is …
Appendix 5—figure 2
Map of the repressor plasmid.
Auxotrophic marker is different depending on the plasmid backbone.
Appendix 5—figure 3
Map of the tagging plasmid.
Resistance marker is NAT or HygR depending on the plasmid number. The colored boxes zoom in to the marked regions to demonstrate how the primers anneal to the plasmid.
Tables
Table 1
Main strains used in this work and their relevant genotype.
A detailed table including all strains used in the figure supplements can be found in Supplementary file 1.
| Y | Name | Relevant genotype |
|---|---|---|
| 70 | autofluorescence | BY4743 derivative, haploid, MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 lys2Δ0 |
| 2683 | PTDH3-const | leu2Δ::PTDH3_citrine-LEU2 |
| 2551 | P2tet-const | leu2Δ::P2tet_citrine-LEU2 |
| 2564 | P3tet-const | leu2Δ::P3tet_citrine-LEU2 |
| 2566 | P5tet-const | leu2Δ::P5tet_citrine-LEU2 |
| 2562 | P2tet-SR | leu2Δ::P2tet_citrine-LEU2 his3Δ::PACT1_tetR-NLS-HIS3 |
| 2573 | P3tet-SR | leu2Δ::P3tet_citrine-LEU2 his3Δ::PACT1_tetR-NLS-HIS3 |
| 2577 | P5tet-SR | leu2Δ::P5tet_citrine-LEU2 his3Δ::PACT1_tetR-NLS-HIS3 |
| 2659 | P5tet.1-const | leu2Δ::P5tet.1_citrine-LEU2 |
| 2656 | P5tet.1-SR | leu2Δ::P5tet.1_citrine-LEU2 his3Δ::PACT1_tetR-NLS-HIS3 |
| 2661 | P7tet.1-const | leu2Δ::P7tet.1_citrine-LEU2 |
| 2663 | P7tet.1-SR | leu2Δ::P7tet.1_citrine-LEU2 his3Δ::PACT1_tetR-NLS-HIS3 |
| 2674 | P7tet.1-AR | leu2Δ::P7tet.1_citrine-LEU2 met15Δ::P7tet.1_tetR-NLS-MET15 |
| 2741 | P7tet.1-cAR | leu2Δ::P7tet.1_citrine-LEU2 met15Δ::P7tet.1_tetR-NLS-MET15 his3Δ::PACT1_tetR-NLS-HIS3 |
| 2673 | P7tet.1-cAR(PACT1-TUP1) | leu2Δ::P7tet.1_citrine-LEU2 his3Δ::PACT1_tetR-NLS-tup1-HIS3 met15Δ::P7tet.1_tetR-NLS-MET15 |
| 2684 | P7tet.1-cAR(PVPH1-TUP1) | leu2Δ::P7tet.1_citrine-LEU2 his3Δ::PVPH1_tetR-NLS-tup1-HIS3 met15Δ::P7tet.1_tetR-NLS-MET15 |
| 2749 | P7tet.1-cAR(PRNR2-TUP1) | leu2Δ::P7tet.1_citrine-LEU2 his3Δ::PRNR2_tetR-NLS-tup1-HIS3 met15Δ::P7tet.1_tetR-NLS-MET15 |
| 2715 | P7tet.1-cAR(P_PREV1-TUP1) | leu2Δ::P7tet.1_citrine-LEU2 his3Δ::P_PREV1_tetR-NLS-tup1-HIS3 met15Δ::P7tet.1_tetR-NLS-MET15 |
| 2669 | P7tet.1-SR(PACT1-TUP1) | leu2Δ::P7tet.1_citrine-LEU2 his3Δ::PACT1_tetR-NLS-tup1-HIS3 |
| 2676 | P7tet.1-SR(PVPH1-TUP1) | leu2Δ::P7tet.1_citrine-LEU2 his3Δ::PVPH1_tetR-NLS-tup1-HIS3 |
| 2717 | P7tet.1-SR(PRNR2-TUP1) | leu2Δ::P7tet.1_citrine-LEU2 his3Δ::PRNR2_tetR-NLS-tup1-HIS3 |
| 2759 | WTC846::citrine | leu2Δ::P7tet.1_citrine-LEU2 ura3Δ::PRNR2_tetR-NLS-tup1_P7tet.1_tetR-NLS-URA3 |
| 2761 | WTC846:: | ura3Δ::PRNR2_tetR-NLS-tup1_P7tet.1_tetR-NLS-URA3 |
| 2769 | parent | whi5Δ::WHI5-mKOkappa-HIS3, myo1Δ::MYO1-mKate(3x)-KanMX, leu2Δ::PRNR2_tetR-NLS-tup1_P7tet.1_tetR-NLS-LEU2 |
| 2772 | WTC846-K1::TOR2 | whi5Δ::WHI5-mKOkappa-HIS3, myo1Δ::MYO1-mKate(3x)-KanMX, leu2Δ::PRNR2_tetR-NLS-tup1_P7tet.1_tetR-NLS-LEU2 P_TOR2::P7tet.1-K1-HygMX |
| 2775 | WTC846-K2::CDC28 | whi5Δ::WHI5-mKOkappa-HIS3, myo1Δ::MYO1-mKate(3x)-KanMX, leu2Δ::PRNR2_tetR-NLS-tup1_P7tet.1_tetR-NLS-LEU2 P_CDC28::P7tet.1-K2-NatMX |
| 2837 | WTC846-K3::CDC20 | whi5Δ::WHI5-mKOkappa-HIS3, myo1Δ::MYO1-mKate(3x)-KanMX, leu2Δ::PRNR2_tetR-NLS-tup1_P7tet.1_tetR-NLS-LEU2 P_CDC20::P7tet.1-K3-NatMX |
| 2788 | WTC846-K2::CDC42 | whi5Δ::WHI5-mKOkappa-HIS3, myo1Δ::MYO1-mKate(3x)-KanMX, leu2Δ::PRNR2_tetR-NLS-tup1_P7tet.1_tetR-NLS-LEU2 P_CDC42::P7tet.1-K2-NatMX |
| 2789 | WTC846-K2::IPL1 | whi5Δ::WHI5-mKOkappa-HIS3, myo1Δ::MYO1-mKate(3x)-KanMX, leu2Δ::PRNR2_tetR-NLS-tup1_P7tet.1_tetR-NLS-LEU2 P_IPL1::P7tet.1-K2-NatMX |
| 2828 | WTC846-K3::PMA1 | whi5Δ::WHI5-mKOkappa-HIS3, myo1Δ::MYO1-mKate(3x)-KanMX, leu2Δ::PRNR2_tetR-NLS-tup1_P7tet.1_tetR-NLS-LEU2 P_PMA1::P7tet.1-K3-NatMX |
| 2773 | WTC846-K3::TOR2 | whi5Δ::WHI5-mKOkappa-HIS3, myo1Δ::MYO1-mKate(3x)-KanMX, leu2Δ::PRNR2_tetR-NLS-tup1_P7tet.1_tetR-NLS-LEU2 P_TOR2::P7tet.1-K3-HygMX |
| 2827 | WTC846-K3::CDC28 | whi5Δ::WHI5-mKOkappa-HIS3, myo1Δ::MYO1-mKate(3x)-KanMX, leu2Δ::PRNR2_tetR-NLS-tup1_P7tet.1_tetR-NLS-LEU2 P_CDC28::P7tet.1-K3-NatMX |
| 2830 | WTC846-K3::PBR1 | whi5Δ::WHI5-mKOkappa-HIS3,myo1Δ::MYO1-mKate(3x)-KanMX, leu2Δ::PRNR2_tetR-NLS-tup1_P7tet.1_tetR-NLS-LEU2 P_PBR1::P7tet.1-K3-NatMX |
| 2849 | WTC846-K3::TPI1 | leu2Δ::PRNR2_tetR-NLS-tup1_P7tet.1_tetR-NLS-LEU2 P_TPI1::P7tet.1-K3-NatMX |
| 2791 | WTC846-K1::WHI5 | whi5Δ::WHI5-mKOkappa-HIS3, myo1Δ::MYO1-mKate(3x)-KanMX, leu2Δ::PRNR2_tetR-NLS-tup1_P7tet.1_tetR-NLS-LEU2 P_WHI5::P7tet.1-K1-NatMX |
| 2929 | WTC846-K1::WHI5(diploid) | BY4743, whi5Δ::WHI5-mKokappa-HIS3/WHI5 myo1Δ::MYO1-mKate(3x)-KanMX/MYO1 leu2Δ::PRNR2_tetR-NLS-tup1_P7tet.1_tetR-NLS-LEU2/leu2Δ0 ura3Δ::PRNR2_tetR-NLS-tup1_P7tet.1_tetR-NLS-URA3/ura3Δ0 P_WHI5::P7tet.1-K1-HygMX/P_WHI5::P7tet.1-K1-NatMX |
Appendix 5—table 1
Repressor plasmids.
| P number | Marker | Backbone pRG number from Gnügge et al., 2016 |
|---|---|---|
| P2365 | URA3 | pRG206 |
| P2370 | LEU2MX | pRG205MX |
| P2371 | HIS3MX | pRG203MX |
| P2372 | LYS2 | pRG207 |
| P2374 | MET15 | pRG201 |
Additional files
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Supplementary file 1
Table S1 Strains used in this study.
Kozak sequence is the last 15 bp before the start codon. Table S2 Plasmids used in this study. * indicates plasmids available through Addgene. These plasmids are sufficient to allow construction of WTC846 strains carrying the cAR architecture without any further construction. They can also be modified to construct strains with genes controlled by the Simple Repression (SR) and Autorepression (AR) architectures presented in this manuscript. Construction of SR strains would require deletion of the negative feedback-controlled TetR from (P2365/2370/2371/2372/2374), and construction of AR strains would require deletion of the constitutively expressed TetR-Tup1 from the same plasmids. Table S3 Oligos used in this study to create strains where WTC846 controls endogenous gene expression. See Appendix 5 for the protocol used for endogenous gene promoter replacement through homology directed repair. These oligos were used in conjunction with P2375 (NatMX) or P2350 (HygMX) to create the linear PCR fragment necessary for promoter replacement, or as colony PCR oligos to confirm correct promoter replacement. Table S4 Sequences used in this study. (*) indicates a shortened t_CYC1 used to avoid homology in plasmids where there are more than one t_CYC1 sequences. (**) indicates the linker sequence used between TetR-nls and the fusion partners MBP and Tup1. (***) indicates the linker sequence used between TetR-nls and the fusion partner GST. Table S5 Parameters used to fit 5-parameter sigmoid curves to experimental data. See Materials and methods for the 5-parameter log logistic forumula.
- https://cdn.elifesciences.org/articles/69549/elife-69549-supp1-v2.tex
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Transparent reporting form
- https://cdn.elifesciences.org/articles/69549/elife-69549-transrepform-v2.docx
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Appendix 1—figure 1—source data 1
Numerical data for Appendix 1—figure 1.
- https://cdn.elifesciences.org/articles/69549/elife-69549-app1-fig1-data1-v2.zip
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Appendix 2—figure 2—source data 1
Numerical data for Appendix 2—figure 2C.
- https://cdn.elifesciences.org/articles/69549/elife-69549-app2-fig2-data1-v2.zip
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Appendix 3—figure 1—source data 1
Numerical data for Appendix 3—figure 1.
- https://cdn.elifesciences.org/articles/69549/elife-69549-app3-fig1-data1-v2.zip
