PMC

Modelling of one-input switches. (A and D) Diagrams showing the genetic circuits for one-input switches without (A) or with (D) the TetR-based delay circuit. In (A), the RDF is expressed from a constitutively active promoter (P_c) only in the LR state. In (D), RDF is expressed from a TetR-repressed promoter (P_tet) in the LR state, and TetR is expressed from a constitutively active promoter (P_c) in the PB state. The input signal is provided by short pulses of arabinose that induce expression of integrase protein (Int) from a separate plasmid (shown between A and D). Int (with or without RDF) provides transitions between the PB (blue) and LR (red) states of the switch DNA. In (D), expression of TetR in the PB state delays RDF expression during the PB→LR transition. (B and C) Modelled kinetics of the PB→LR (B) and LR→PB (C) transitions for the switch without TetR. (E and F) Modelled kinetics of the PB→LR (E) and LR→PB (F) transitions for the switch with TetR controlled by the switch. Graphs show the relative amounts (arbitrary units; AU) of LR (red), PB (blue), arabinose (purple) and the concentrations (μM) of Int (green), RDF (black) and TetR (cyan). A single 12-min arabinose pulse was given at time point 0.5 h.

Repeated operation of a switch with state-dependent expression of RDF. (A) Escherichia coli DS941 Z1, which has a chromosomal copy of the tetracycline repressor gene, was co-transformed with a plasmid expressing φC31 integrase from the P_BAD promoter (pBAD-INT) and pSWITCH2*. φC31 RDF is expressed from P_LtetO-1 (P_TET) and an optimized RBS (yellow oval) on pSWITCH2*, but only when aTc is present and the switch is in the LR state. In the PB state, arabinose induces expression of integrase leading to the conversion of PB→LR. In the LR state, addition of arabinose and aTc induces integrase and RDF, converting LR→PB. (B) Starting from the PB state, DS941 Z1 containing pSWITCH2* and pBAD-INT was subjected to five cycles of induction first with arabinose and then with arabinose and aTc. For each induction, cells from the previous stage were diluted 1:1000 and then grown overnight to stationary phase with either 0.2% arabinose or 0.2% arabinose plus 100 ng/ml aTc. Plasmid DNA was isolated and cleaved with NheI before agarose gel electrophoresis. The percentage of pSWITCH2* DNA in the LR state is shown below each lane. GFP fluorescence scans of cell cultures at each stage are shown aligned with the corresponding lanes of the gel above.

Directional recombination on high copy-number plasmids. (A) Diagrams of high copy-number switch plasmids containing an invertible DNA segment in the PB (pSWITCH0-PB) or the LR state (pSWITCH0-LR). (B) These plasmids were introduced into Escherichia coli DS941 together with a plasmid expressing either φC31 integrase (pBAD-INT; left panel) or integrase with its RDF (pBAD[INT+RDF]; right panel) from the arabinose-inducible P_BAD promoter. Cells were grown overnight in LB containing 0.2% glucose to repress expression from P_BAD (lanes 1 and 4), or overnight with 0.2% arabinose to induce expression from P_BAD (lanes 2 and 5). The cells that had been grown overnight with arabinose were subsequently diluted 1:1000 and grown overnight again without inducer (lanes 3 and 6). Plasmid DNA was purified and cut with AlwNI and EcoRV. (C) The left panel (lanes 1–3) shows the same samples as shown in the left panel of (B), run on an agarose gel without prior restriction digestion, to reveal plasmid multimers produced by intermolecular recombination. A multimer resolution site (cer) was added to pSWITCH0 at the position indicated in (A) (dotted lines) to create pSWITCH0-cer. DS941 containing pSWITCH0-PB-cer and pBAD-INT was grown with or without arabinose induction as in (B) and analysed by gel electrophoresis without restriction digestion (lanes 4–6).

Segregation of pSWITCH3* in PB and LR states. (A) Cells containing pBAD-INT and pSWITCH3* in the LR state were subjected to a pulse of arabinose to induce switching to the PB state. Cells were diluted 1000-fold and then grown to stationary phase (10 generations) a total of seven times. At each stage, GFP fluorescence of ∼30 000 individual cells was measured by flow cytometry. (B and C) Stochastic simulation of cell fluorescence and plasmid segregation over 70 generations. The simulation was started with a population of 3000 cells, all with 16 PB plasmid copies and 4 LR copies (20% LR). In the model, plasmids replicate and then are randomly distributed between daughter cells at cell division. Each cell produces GFP at a rate proportional to the LR copy-number and inherits half the GFP content of its mother cell. The left panels show simulated flow cytometry plots at 0, 10, 30 and 70 generations. The number of cells in each fluorescence bin is plotted. Right panels show the distribution of LR copy-number in cells at 0, 10, 30 and 70 generations.

Repeated operation of the pSWITCH3* single-input binary counting module. (A) The state-based switch (pSWITCH2) was converted to a single-input binary counting module pSWITCH3 by adding a delay circuit consisting of the tetR gene expressed from a constitutive promoter only in the PB state. The ribosome-binding site for the rdf gene was then optimized for switching in both directions to give pSWITCH3*. (B and C) Operation of pSWITCH3* over eight cycles of pulsed integrase expression starting from the PB state. For each cycle, exponentially growing cells were exposed to 0.2% arabinose for 15 min to induce integrase expression, followed by 1:1000 dilution into media containing 0.2% glucose to repress further expression. (B) Plasmid DNA was purified and analysed by restriction digestion and agarose gel electrophoresis. The percentage of DNA in the LR state is shown below in each lane. (C) GFP fluorescence of approximately 30 000 cells was measured by flow cytometry after each overnight culture. Percentages of cells in high, intermediate and low fluorescence states are shown on the plots, with gates shown as red dashed lines.

Repeated operation of pSWITCH3* allowing 70 generations for plasmid segregation between arabinose pulses. (A) Cells containing pBAD-INT and pSWITCH3*-PB were exposed to eight arabinose pulses, each pulse followed by 70 generations to allow almost complete plasmid segregation. Histograms show fluorescence of ∼30 000 cells measured by flow cytometry after each pulse and 70 generations of growth. (B) Graph showing results of three repeats of the same experiment, starting from either pSWITCH3*-PB (blue circles) or pSWITCH3*-LR (orange circles). The percentage of cells with high GFP fluorescence is plotted. Values are the average from three replicates, and error bars represent the standard deviation. Lines show predictions from a model in which 95% of all cells in the PB state change to LR, and 85% of all cells in the LR state change to PB at each pulse. (C) Plasmid DNA was isolated from the samples shown in (A), cleaved with SpeI and analysed by agarose gel electrophoresis. Fluorescence scans of 200 μl of cell culture at each stage are shown aligned with the corresponding lanes of the gel above. (D) Quantitation of DNA in PB and LR states, starting from either PB (blue line) or LR (orange dashed line).

Repeated operation of a low copy-number inversion switch by inducible expression of integrase and RDF. (A) pSWITCH1 has a low copy-number pSC101 origin and a cer site and carries φC31 att sites flanking a constitutively active promoter (arrow). RFP is expressed in the PB state, while GFP is expressed in the LR state. (B) pSWITCH1-PB was introduced into Escherichia coli DS941 cells together with pBAD-INT and either pTET[INT+RDF] expressing integrase and RDF as two separate proteins (left), or pTET[INT+RDF]^FUS expressing the Int-RDF fusion protein (right) under the control of the aTc inducible P_LtetO-1 promoter (P_TET). Cells were grown overnight in 0.2% glucose (lane 1 on each gel). Cultures were diluted 1:40 into fresh LB and grown for 90 min. Arabinose was then added, and cells were grown for a further 30 min before 1:1000 dilution and overnight growth in fresh media containing 0.2% glucose. This treatment constitutes an arabinose pulse (ara). This was followed by similar aTc, then arabinose, then aTc pulses. DNA was isolated from each overnight culture, cleaved with XhoI and analysed by agarose gel electrophoresis. The percentage of pSWITCH1 in the LR state is shown beneath each lane. GFP and RFP fluorescence scans of 200 μl of cell suspension in 96-well plates after each overnight culture are shown aligned with the corresponding lanes of the gels.

Inversion switches for binary memory and counters. (A) The integrase (Int) protein on its own catalyses recombination between DNA sites attP and attB to create two new sites, attL and attR. Recombination between attL and attR only occurs if the recombination directionality factor (RDF) and integrase are both present. If att sites are in inverted repeat on either side of a promoter, recombination will reverse the orientation of the promoter and control transcription of a gene outside the invertible DNA segment (e.g. the gene for green fluorescent protein (GFP)). The two states of the inversion switch can be thought of as representing the binary digits 0 and 1. (B) Expression of the RDF can be placed under the control of the switch so that is it expressed in the LR state but not the PB state. A pulse of integrase expression should toggle the state of the switch from PB→LR or LR→PB. (C) Three inversion switches based on orthogonal integrases can be used to represent any binary number from 0 to 111 (0 to 7 decimal). The state of each switch is output through expression of three different fluorescent proteins, exemplified here as green-, red- and cyan fluorescent proteins (GFP, RFP and CFP). The figure shows the design for a binary ‘ripple’ counter that will step sequentially through all binary numbers between 000 and 111 in response to a repeated input signal. Each input signal (black arrows) toggles the state of the ‘units’ (GFP) switch so that it alternates between PB (0) and LR (1) states. As the units switch changes from 1 to 0, it sends a signal (green arrows) to toggle the ‘twos’ (RFP) switch, which in turn sends a signal (red arrows) to toggle the ‘fours’ (CFP) switch as it changes from 1 to 0.