Engineering of a synthetic quadrastable gene network to approach Waddington landscape and cell fate determination
- Arizona State University, United States
- King’s College, University of Aberdeen, United Kingdom
Figures
Figure 1 with 1 supplement
Conceptual and experimental design of MINPA and its sub-networks.
(A) Abstract diagram of MINPA topology, where X and Y mutually inhibit (T-bars) each other and auto-activate (arrowheads) itself. Four inducers to regulate the four color-coded regulatory edges are …
Figure 1—figure supplement 1
Experimental design, topological hierarchy and multistability probability analysis of MINPA sub-networks.
(A) Abstract diagrams and molecular implementation of the eight MINPA sub-network topologies, including tunable positive feedbacks (T6 and T9), mutual inhibition (T5), dual-positive feedbacks (T10), …
Figure 2 with 3 supplements
Sequential induction of MINPA and its sub-networks.
(A) Schematic illustration of rationale for sequential induction. This two-parameter bifurcation diagram of a bistable toggle-switch depicts all steady state values of response (Z-axis) with …
Figure 2—figure supplement 1
Experimental design and validation of sequential induction strategy in a synthetic toggle switch circuit.
(A) Abstract diagram and molecular implementation of the toggle switch circuit. TetR (R) and LacI (I) mutually inhibit each other through binding to Ptet and Plac promoter, respectively. IPTG and …
Figure 2—figure supplement 2
Time course results of sequential induction for the MINPA (T15) circuit.
(A) Arabinose (Ara) and IPTG were sequentially (left and middle columns) or simultaneously (right column) applied to induce T15. The first inducer was applied for 5 hr, and then the second inducer …
Figure 2—figure supplement 3
Sequential induction for circuits T5, T7, T13, and T15 with inducers IPTG and aTc.
Left: IPTG was first applied to induce the circuits, and then aTc was added; Middle: aTc was first applied to induce the circuits, and then IPTG was added; Right: IPTG and aTc were added …
Figure 3 with 2 supplements
Bifurcation analysis and hysteresis of MINPA.
(A) Engineered MINPA is tunable to reach four individual states: low-low, GFP, mCherry, and high-high, under no induction, 1*10−4 M AHL and 100 ng/ml aTc, 2.5*10−5 (m/v) Arabinose, 1*10−4 M AHL and …
Figure 3—figure supplement 1
Another view of the 3-D bifurcation diagram of MINPA at C2.
The circuit’s quadrastability is illustrated as four similar-sized colored spheres on the same gray plane, which represents the low-low, GFP, mCherry, and high-high state, respectively. Blue lines …
Figure 3—figure supplement 2
Bifurcation analysis for and hysteresis of MINPA with induction of Arabinose and AHL.
(A–D) One-dimensional bifurcation analysis for all four inducers in MINPA. We perform bifurcation analysis for each inducer while setting the concentration of other inducers to be very small (10−10).…
Figure 4 with 1 supplement
Model-guided quadrastability of MINPA through triple induction.
(A) Dynamic evolution of computed energy landscapes of MINPA under sequential/simultaneous inductions of Arabinose, and/or AHL and aTc. Center route: simultaneous induction with three inducers; Left …
Figure 4—figure supplement 1
Cells’ states under induction with the first inducer, microfludic results to demonstrate quadrastability with IPTG and aTc induction, and time course of sequential induction of AHL, aTc and Ara.
(A) Up left: Flow cytometry result for cells simultaneously induced with 2.5*10−5m/v Arabinose, 1*10−4 M AHL and 200 ng/ml aTc for 6.5 hr. Up right: Cells were first induced with 1*10−4 M and 400 …
Appendix 1—figure 1
Cell growth rates under each inducer and inducer combinations.
Growth curves for the cells under the four individual inducers: Ara; AHL; aTc; IPTG, and inducer combinations: AHL and aTc; AHL, aTc, and Ara. Ara: 2.5*10−5m/v; AHL: 1*10−5 M, aTc: 200 ng/ml, IPTG: …
Videos
Appendix 1—Video 1
A time-lapse movie growing in the microfluidic chip for 24 hr.
Time course of the cells growing and fluorescence state change with 2*10−4 M IPTG and 200 ng/ml aTc induction in the trap. The red flow is medium without inducer for 6 hr, and then cells switch to …
Tables
Table 1
Components from the Registry of standard biological parts
| Biobrick number | Abbreviation in the paper | Description |
|---|---|---|
| BBa_C0080 | AraC | AraC arabinose operon regulatory protein from E. coli |
| BBa_C0040 | TetR | Tetracycline repressor from transposon Tn10 |
| BBa_C0062 | LuxR | LuxR activator from Aliivibrio fischeri |
| BBa_C0012 | LacI | LacI repressor from E. coli |
| BBa_E0240 | GFP | GFP generator |
| BBa_J06702 | mCherry | RFP generator |
| BBa_K176002 | Plux/tet | Hybrid promoter with LuxR/HSL- and TetR-binding sites |
| BBa_B0034 | RBS | Ribosome binding site |
| BBa_B0015 | Terminator | Transcriptional terminator (double) |
| BBa_K176009 | CP | Constitutive promoter |
| pSB1K3 | pSB1K3 | High copy BioBrick assembly plasmid with kanamycin resistance |
| pSB1A3 | pSB1A3 | High copy BioBrick assembly plasmid with ampicillin resistance |
Additional files
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Source code file 1
Flow cytometry data analysis file.
- https://doi.org/10.7554/eLife.23702.015
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Source code file 2
Bifurcation analysis in Figure 3.
- https://doi.org/10.7554/eLife.23702.016
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Source code file 3
Potential landscape calculation file.
- https://doi.org/10.7554/eLife.23702.017
