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(A) Architecture of the recombinase-based memory switch. The input module (pSwitch) contains IPTG- and arabinose-inducible promoters driving the expression of HbiF and FimE, respectively. The output module (pReporter) contains recombinase recognition sites flanking a constitutive promoter (Ptrc*) controlling RFP and GFP expression. Site-specific <t>DNA</t> inversion between the inverted repeats (IRL/IRR) reconfigures the recombination sites, reversing the promoter orientation and thereby toggling expression between RFP and GFP . (B) Flow cytometry analysis of cells initialized in the Green-On state and analyzed on day 3. The percentages of Red, uncommitted, and Green cells are reported as indicated. Red circles indicate the effect of recombinase leakiness and non-orthogonality. (C) Flow cytometry analysis of cells initialized in the Green-On state and switched to the Red-On state on day 3. (D) Flow cytometry analysis of cells initialized in the Red-On state and analyzed on day 3. (E) Flow cytometry analysis of cells initialized in the Red-On state and switched to the Green-On state on day 3.
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(A) Architecture of the recombinase-based memory switch. The input module (pSwitch) contains IPTG- and arabinose-inducible promoters driving the expression of HbiF and FimE, respectively. The output module (pReporter) contains recombinase recognition sites flanking a constitutive promoter (Ptrc*) controlling RFP and GFP expression. Site-specific <t>DNA</t> inversion between the inverted repeats (IRL/IRR) reconfigures the recombination sites, reversing the promoter orientation and thereby toggling expression between RFP and GFP . (B) Flow cytometry analysis of cells initialized in the Green-On state and analyzed on day 3. The percentages of Red, uncommitted, and Green cells are reported as indicated. Red circles indicate the effect of recombinase leakiness and non-orthogonality. (C) Flow cytometry analysis of cells initialized in the Green-On state and switched to the Red-On state on day 3. (D) Flow cytometry analysis of cells initialized in the Red-On state and analyzed on day 3. (E) Flow cytometry analysis of cells initialized in the Red-On state and switched to the Green-On state on day 3.
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(A) Architecture of the recombinase-based memory switch. The input module (pSwitch) contains IPTG- and arabinose-inducible promoters driving the expression of HbiF and FimE, respectively. The output module (pReporter) contains recombinase recognition sites flanking a constitutive promoter (Ptrc*) controlling RFP and GFP expression. Site-specific <t>DNA</t> inversion between the inverted repeats (IRL/IRR) reconfigures the recombination sites, reversing the promoter orientation and thereby toggling expression between RFP and GFP . (B) Flow cytometry analysis of cells initialized in the Green-On state and analyzed on day 3. The percentages of Red, uncommitted, and Green cells are reported as indicated. Red circles indicate the effect of recombinase leakiness and non-orthogonality. (C) Flow cytometry analysis of cells initialized in the Green-On state and switched to the Red-On state on day 3. (D) Flow cytometry analysis of cells initialized in the Red-On state and analyzed on day 3. (E) Flow cytometry analysis of cells initialized in the Red-On state and switched to the Green-On state on day 3.
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(A) 1.2% agarose gel electrophoresis of the PCR fragment and the digested pB40-79-PTasRNA lon vector. MW = 1 kb Plus <t>DNA</t> ladder; 1 = asRNA repB PCR fragment (110 bp); 2 = Xho I-pB40-79-PT- Pst I linear fragment (4,979 bp); 3 = pB40-79-PTasRNA lon plasmid DNA (negative control). ( B ) Map of the PTasRNA vector. The diagram illustrates the modular organization of the silencing vector. The cloning site designed for the antisense <t>sequence</t> (pink) is flanked by Pst I and Xho I restriction sites located between the two paired termini sequences (in gray). The vector includes two origins of replication: oriC for the propagation of the plasmid in E. coli strains (in red) and oriR for propagation in Ph TAC125 (in green); oriT is pivotal for the conjugative transfer (in orange); Ph LacR encodes the regulator of p LacZ promoter (not shown); amp R represents the ampicillin resistance marker (in blue).
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(A) 1.2% agarose gel electrophoresis of the PCR fragment and the digested pB40-79-PTasRNA lon vector. MW = 1 kb Plus <t>DNA</t> ladder; 1 = asRNA repB PCR fragment (110 bp); 2 = Xho I-pB40-79-PT- Pst I linear fragment (4,979 bp); 3 = pB40-79-PTasRNA lon plasmid DNA (negative control). ( B ) Map of the PTasRNA vector. The diagram illustrates the modular organization of the silencing vector. The cloning site designed for the antisense <t>sequence</t> (pink) is flanked by Pst I and Xho I restriction sites located between the two paired termini sequences (in gray). The vector includes two origins of replication: oriC for the propagation of the plasmid in E. coli strains (in red) and oriR for propagation in Ph TAC125 (in green); oriT is pivotal for the conjugative transfer (in orange); Ph LacR encodes the regulator of p LacZ promoter (not shown); amp R represents the ampicillin resistance marker (in blue).
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(A) 1.2% agarose gel electrophoresis of the PCR fragment and the digested pB40-79-PTasRNA lon vector. MW = 1 kb Plus <t>DNA</t> ladder; 1 = asRNA repB PCR fragment (110 bp); 2 = Xho I-pB40-79-PT- Pst I linear fragment (4,979 bp); 3 = pB40-79-PTasRNA lon plasmid DNA (negative control). ( B ) Map of the PTasRNA vector. The diagram illustrates the modular organization of the silencing vector. The cloning site designed for the antisense <t>sequence</t> (pink) is flanked by Pst I and Xho I restriction sites located between the two paired termini sequences (in gray). The vector includes two origins of replication: oriC for the propagation of the plasmid in E. coli strains (in red) and oriR for propagation in Ph TAC125 (in green); oriT is pivotal for the conjugative transfer (in orange); Ph LacR encodes the regulator of p LacZ promoter (not shown); amp R represents the ampicillin resistance marker (in blue).
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(A) 1.2% agarose gel electrophoresis of the PCR fragment and the digested pB40-79-PTasRNA lon vector. MW = 1 kb Plus <t>DNA</t> ladder; 1 = asRNA repB PCR fragment (110 bp); 2 = Xho I-pB40-79-PT- Pst I linear fragment (4,979 bp); 3 = pB40-79-PTasRNA lon plasmid DNA (negative control). ( B ) Map of the PTasRNA vector. The diagram illustrates the modular organization of the silencing vector. The cloning site designed for the antisense <t>sequence</t> (pink) is flanked by Pst I and Xho I restriction sites located between the two paired termini sequences (in gray). The vector includes two origins of replication: oriC for the propagation of the plasmid in E. coli strains (in red) and oriR for propagation in Ph TAC125 (in green); oriT is pivotal for the conjugative transfer (in orange); Ph LacR encodes the regulator of p LacZ promoter (not shown); amp R represents the ampicillin resistance marker (in blue).
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(A) 1.2% agarose gel electrophoresis of the PCR fragment and the digested pB40-79-PTasRNA lon vector. MW = 1 kb Plus <t>DNA</t> ladder; 1 = asRNA repB PCR fragment (110 bp); 2 = Xho I-pB40-79-PT- Pst I linear fragment (4,979 bp); 3 = pB40-79-PTasRNA lon plasmid DNA (negative control). ( B ) Map of the PTasRNA vector. The diagram illustrates the modular organization of the silencing vector. The cloning site designed for the antisense <t>sequence</t> (pink) is flanked by Pst I and Xho I restriction sites located between the two paired termini sequences (in gray). The vector includes two origins of replication: oriC for the propagation of the plasmid in E. coli strains (in red) and oriR for propagation in Ph TAC125 (in green); oriT is pivotal for the conjugative transfer (in orange); Ph LacR encodes the regulator of p LacZ promoter (not shown); amp R represents the ampicillin resistance marker (in blue).
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(A) Architecture of the recombinase-based memory switch. The input module (pSwitch) contains IPTG- and arabinose-inducible promoters driving the expression of HbiF and FimE, respectively. The output module (pReporter) contains recombinase recognition sites flanking a constitutive promoter (Ptrc*) controlling RFP and GFP expression. Site-specific DNA inversion between the inverted repeats (IRL/IRR) reconfigures the recombination sites, reversing the promoter orientation and thereby toggling expression between RFP and GFP . (B) Flow cytometry analysis of cells initialized in the Green-On state and analyzed on day 3. The percentages of Red, uncommitted, and Green cells are reported as indicated. Red circles indicate the effect of recombinase leakiness and non-orthogonality. (C) Flow cytometry analysis of cells initialized in the Green-On state and switched to the Red-On state on day 3. (D) Flow cytometry analysis of cells initialized in the Red-On state and analyzed on day 3. (E) Flow cytometry analysis of cells initialized in the Red-On state and switched to the Green-On state on day 3.

Journal: bioRxiv

Article Title: Engineering Persistent and Rewritable Genetic Memory in Bacteria through Multiscale Plasmid Competition

doi: 10.64898/2026.09.18.752696

Figure Lengend Snippet: (A) Architecture of the recombinase-based memory switch. The input module (pSwitch) contains IPTG- and arabinose-inducible promoters driving the expression of HbiF and FimE, respectively. The output module (pReporter) contains recombinase recognition sites flanking a constitutive promoter (Ptrc*) controlling RFP and GFP expression. Site-specific DNA inversion between the inverted repeats (IRL/IRR) reconfigures the recombination sites, reversing the promoter orientation and thereby toggling expression between RFP and GFP . (B) Flow cytometry analysis of cells initialized in the Green-On state and analyzed on day 3. The percentages of Red, uncommitted, and Green cells are reported as indicated. Red circles indicate the effect of recombinase leakiness and non-orthogonality. (C) Flow cytometry analysis of cells initialized in the Green-On state and switched to the Red-On state on day 3. (D) Flow cytometry analysis of cells initialized in the Red-On state and analyzed on day 3. (E) Flow cytometry analysis of cells initialized in the Red-On state and switched to the Green-On state on day 3.

Article Snippet: DNA sequencing used Plasmidsaurus DNA whole plasmid sequencing service.

Techniques: Expressing, Flow Cytometry, Analysis

(A) 1.2% agarose gel electrophoresis of the PCR fragment and the digested pB40-79-PTasRNA lon vector. MW = 1 kb Plus DNA ladder; 1 = asRNA repB PCR fragment (110 bp); 2 = Xho I-pB40-79-PT- Pst I linear fragment (4,979 bp); 3 = pB40-79-PTasRNA lon plasmid DNA (negative control). ( B ) Map of the PTasRNA vector. The diagram illustrates the modular organization of the silencing vector. The cloning site designed for the antisense sequence (pink) is flanked by Pst I and Xho I restriction sites located between the two paired termini sequences (in gray). The vector includes two origins of replication: oriC for the propagation of the plasmid in E. coli strains (in red) and oriR for propagation in Ph TAC125 (in green); oriT is pivotal for the conjugative transfer (in orange); Ph LacR encodes the regulator of p LacZ promoter (not shown); amp R represents the ampicillin resistance marker (in blue).

Journal: Bio-protocol

Article Title: Plasmid Curing of Pseudoalteromonas haloplanktis TAC125 Using Homologous Recombination and PTasRNA Gene Silencing

doi: 10.21769/BioProtoc.5687

Figure Lengend Snippet: (A) 1.2% agarose gel electrophoresis of the PCR fragment and the digested pB40-79-PTasRNA lon vector. MW = 1 kb Plus DNA ladder; 1 = asRNA repB PCR fragment (110 bp); 2 = Xho I-pB40-79-PT- Pst I linear fragment (4,979 bp); 3 = pB40-79-PTasRNA lon plasmid DNA (negative control). ( B ) Map of the PTasRNA vector. The diagram illustrates the modular organization of the silencing vector. The cloning site designed for the antisense sequence (pink) is flanked by Pst I and Xho I restriction sites located between the two paired termini sequences (in gray). The vector includes two origins of replication: oriC for the propagation of the plasmid in E. coli strains (in red) and oriR for propagation in Ph TAC125 (in green); oriT is pivotal for the conjugative transfer (in orange); Ph LacR encodes the regulator of p LacZ promoter (not shown); amp R represents the ampicillin resistance marker (in blue).

Article Snippet: DNA sequencing service (e.g., Eurofins Genomics, Ebersberg, Germany) 7.

Techniques: Agarose Gel Electrophoresis, Plasmid Preparation, Negative Control, Cloning, Sequencing, Marker