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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) Corallimorpharian mucus collection. After initial salt water dripped off animals and plates, mucus was collected from adult Corallimorpharian animals by tilting over 1.5 mL centrifuge tubes and collecting the run-off. B,C) R. florida mucus exhibit consistently higher CFU counts than those that harbored seawater. Corallimorph mucus (A) and surrounding sea water (C) were cultured on Marine Agar plates and the number of colony forming units were quantified. ( D) Corallimorpharian mucus harbors morphologically diverse bacterial isolates. Colonies isolated from R. florida mucus exhibited substantial variation in pigmentation and morphology, including dark purple/black (A), translucent cream (B-D), white (E), and peach (F) morphotypes. Isolates also differed in colony architecture and growth characteristics. Among these, one isolate displayed an unusual agar-pitting phenotype (G), suggesting extracellular agar-degrading activity. ( E,F) R. florida were infected with A2 Vibrio . Clonal polyps from control treatment (E) and high bacterial exposure (F) were pooled in equal quantities for mRNA library construction and <t>sequencing</t> using MiSeq technology (Illumina).
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(A) Corallimorpharian mucus collection. After initial salt water dripped off animals and plates, mucus was collected from adult Corallimorpharian animals by tilting over 1.5 mL centrifuge tubes and collecting the run-off. B,C) R. florida mucus exhibit consistently higher CFU counts than those that harbored seawater. Corallimorph mucus (A) and surrounding sea water (C) were cultured on Marine Agar plates and the number of colony forming units were quantified. ( D) Corallimorpharian mucus harbors morphologically diverse bacterial isolates. Colonies isolated from R. florida mucus exhibited substantial variation in pigmentation and morphology, including dark purple/black (A), translucent cream (B-D), white (E), and peach (F) morphotypes. Isolates also differed in colony architecture and growth characteristics. Among these, one isolate displayed an unusual agar-pitting phenotype (G), suggesting extracellular agar-degrading activity. ( E,F) R. florida were infected with A2 Vibrio . Clonal polyps from control treatment (E) and high bacterial exposure (F) were pooled in equal quantities for mRNA library construction and <t>sequencing</t> using MiSeq technology (Illumina).
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(A) Corallimorpharian mucus collection. After initial salt water dripped off animals and plates, mucus was collected from adult Corallimorpharian animals by tilting over 1.5 mL centrifuge tubes and collecting the run-off. B,C) R. florida mucus exhibit consistently higher CFU counts than those that harbored seawater. Corallimorph mucus (A) and surrounding sea water (C) were cultured on Marine Agar plates and the number of colony forming units were quantified. ( D) Corallimorpharian mucus harbors morphologically diverse bacterial isolates. Colonies isolated from R. florida mucus exhibited substantial variation in pigmentation and morphology, including dark purple/black (A), translucent cream (B-D), white (E), and peach (F) morphotypes. Isolates also differed in colony architecture and growth characteristics. Among these, one isolate displayed an unusual agar-pitting phenotype (G), suggesting extracellular agar-degrading activity. ( E,F) R. florida were infected with A2 Vibrio . Clonal polyps from control treatment (E) and high bacterial exposure (F) were pooled in equal quantities for mRNA library construction and <t>sequencing</t> using MiSeq technology (Illumina).
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HeLa:Cas9 cells in 10cm plates were transfected with overexpression constructs for HPV16L2, encoding a C-terminal HA tag, and either an overexpression construct for PLCδ4 or PLCγ1, both containing the <t>sequence</t> for both C-terminal FLAG tag and c-Myc tag, or pCMV6, an empty vector control. Transfected cells were harvested 24h post-transfection, lysed in buffer containing 1% NP40 and 0.1% SDS, and then (cleared) lysates were incubated with magnetic beads coupled to anti-HA antibodies to pull down L2. Co-immunoprecipitation (CoIP) of PLCδ4 in bead elutions (“Elutes”) was determined using Western blot. Western blotting against the HA and c-Myc tags was used to confirm the presence of L2, PLCδ4, and PLCγ1 in “elutes” and “cleared lysates”. Blots are representative of three independent experiments ( N =3).
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HeLa:Cas9 cells in 10cm plates were transfected with overexpression constructs for HPV16L2, encoding a C-terminal HA tag, and either an overexpression construct for PLCδ4 or PLCγ1, both containing the <t>sequence</t> for both C-terminal FLAG tag and c-Myc tag, or pCMV6, an empty vector control. Transfected cells were harvested 24h post-transfection, lysed in buffer containing 1% NP40 and 0.1% SDS, and then (cleared) lysates were incubated with magnetic beads coupled to anti-HA antibodies to pull down L2. Co-immunoprecipitation (CoIP) of PLCδ4 in bead elutions (“Elutes”) was determined using Western blot. Western blotting against the HA and c-Myc tags was used to confirm the presence of L2, PLCδ4, and PLCγ1 in “elutes” and “cleared lysates”. Blots are representative of three independent experiments ( N =3).
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HeLa:Cas9 cells in 10cm plates were transfected with overexpression constructs for HPV16L2, encoding a C-terminal HA tag, and either an overexpression construct for PLCδ4 or PLCγ1, both containing the <t>sequence</t> for both C-terminal FLAG tag and c-Myc tag, or pCMV6, an empty vector control. Transfected cells were harvested 24h post-transfection, lysed in buffer containing 1% NP40 and 0.1% SDS, and then (cleared) lysates were incubated with magnetic beads coupled to anti-HA antibodies to pull down L2. Co-immunoprecipitation (CoIP) of PLCδ4 in bead elutions (“Elutes”) was determined using Western blot. Western blotting against the HA and c-Myc tags was used to confirm the presence of L2, PLCδ4, and PLCγ1 in “elutes” and “cleared lysates”. Blots are representative of three independent experiments ( N =3).
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HeLa:Cas9 cells in 10cm plates were transfected with overexpression constructs for HPV16L2, encoding a C-terminal HA tag, and either an overexpression construct for PLCδ4 or PLCγ1, both containing the <t>sequence</t> for both C-terminal FLAG tag and c-Myc tag, or pCMV6, an empty vector control. Transfected cells were harvested 24h post-transfection, lysed in buffer containing 1% NP40 and 0.1% SDS, and then (cleared) lysates were incubated with magnetic beads coupled to anti-HA antibodies to pull down L2. Co-immunoprecipitation (CoIP) of PLCδ4 in bead elutions (“Elutes”) was determined using Western blot. Western blotting against the HA and c-Myc tags was used to confirm the presence of L2, PLCδ4, and PLCγ1 in “elutes” and “cleared lysates”. Blots are representative of three independent experiments ( N =3).
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Step 1 ) The sensors are printed with biodegradable PHBV, poly(3-hydroxybutuyrate- co -3-hydroxyvalerate), on one side and non-biodegradable PMMA, poly(methyl methacrylate) on the other side as a control. Step 2 ) UV-sterilized sensors are inserted into soil mesocosms and monitor PHBV degradation in real-time as an increase in resistance, which we normalize to the non-degraded PMMA. Step 3 ) At the end of the mesocosm incubation, the sensors are removed and we can summarise the PHBV degradation signal to identify which soils have the highest capacity to degrade PHBV. Step 4 ) PHBV and PMMA material from sensors incubated in the highest PHBV-degrading soils are scraped off and used as starting material for culture-independent ( Step 5 , left panel) and culture-dependent ( Step 6 , right panel) approaches to identify novel PHBV degraders. Step 5, left ) We used the PHBV and PMMA material from high degrading soils for 16S rRNA and ITS marker gene <t>sequencing</t> to identify microbes enriched on degraded PHBV compared to non-degraded PMMA from each in situ sensor. Step 5, right ) We also used the PHBV material from high degrading soils to isolate novel PHBV degraders on agar plates with a PHBV overlay (inset). Isolates that formed clearing zones on plates were cultured in liquid media to measure their continued capacity to degrade PHBV. We also measured the respiration rates of isolates growing with PHBV as their sole carbon source to confirm PHBV catabolism.
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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) Corallimorpharian mucus collection. After initial salt water dripped off animals and plates, mucus was collected from adult Corallimorpharian animals by tilting over 1.5 mL centrifuge tubes and collecting the run-off. B,C) R. florida mucus exhibit consistently higher CFU counts than those that harbored seawater. Corallimorph mucus (A) and surrounding sea water (C) were cultured on Marine Agar plates and the number of colony forming units were quantified. ( D) Corallimorpharian mucus harbors morphologically diverse bacterial isolates. Colonies isolated from R. florida mucus exhibited substantial variation in pigmentation and morphology, including dark purple/black (A), translucent cream (B-D), white (E), and peach (F) morphotypes. Isolates also differed in colony architecture and growth characteristics. Among these, one isolate displayed an unusual agar-pitting phenotype (G), suggesting extracellular agar-degrading activity. ( E,F) R. florida were infected with A2 Vibrio . Clonal polyps from control treatment (E) and high bacterial exposure (F) were pooled in equal quantities for mRNA library construction and sequencing using MiSeq technology (Illumina).

Journal: bioRxiv

Article Title: Comparative genomics and microbiome profiling reveal a conserved Vibrio rich mucus microbiota of the Florida false coral ( Ricordea florida )

doi: 10.64898/2026.09.17.752451

Figure Lengend Snippet: (A) Corallimorpharian mucus collection. After initial salt water dripped off animals and plates, mucus was collected from adult Corallimorpharian animals by tilting over 1.5 mL centrifuge tubes and collecting the run-off. B,C) R. florida mucus exhibit consistently higher CFU counts than those that harbored seawater. Corallimorph mucus (A) and surrounding sea water (C) were cultured on Marine Agar plates and the number of colony forming units were quantified. ( D) Corallimorpharian mucus harbors morphologically diverse bacterial isolates. Colonies isolated from R. florida mucus exhibited substantial variation in pigmentation and morphology, including dark purple/black (A), translucent cream (B-D), white (E), and peach (F) morphotypes. Isolates also differed in colony architecture and growth characteristics. Among these, one isolate displayed an unusual agar-pitting phenotype (G), suggesting extracellular agar-degrading activity. ( E,F) R. florida were infected with A2 Vibrio . Clonal polyps from control treatment (E) and high bacterial exposure (F) were pooled in equal quantities for mRNA library construction and sequencing using MiSeq technology (Illumina).

Article Snippet: Some isolates were re-sequenced using the Plasmidsaurus Whole Genome Sequencing service (Oxford Nanopore, R10.4.1 long-reads and de novo genome assembly).

Techniques: Cell Culture, Isolation, Morphology, Cream, Activity Assay, Infection, Control, Sequencing

A. Relative abundance (%) of bacteria Genera collected from polyps from distinct tank systems reveals a dominance of Vibrio . Columns of each bar graph represent a polyp (A or B) and tank collected from is noted with the number. B. Polyps from the same tank show no difference in bacterial assemblage. A principal component analysis (PCA) reveals no differences in structure of bacterial communities between tank systems (12, 14, and 15 represented by blue, green and purple circles). X and Y axes show Principal Component 1 and Principal Component 2 that explain 56% and 22% of the total variance, respectively. (C). Shared and unique ASVs among corallimorph-associated bacterial communities. UpSet plot showing the overlap of amplicon sequence variants (ASVs) among Tanks 12, 14, and 15. An ASV was considered present within a tank only if it was detected in both animals sampled from that tank. Numbers indicate ASVs unique to individual tanks or shared among multiple tanks.

Journal: bioRxiv

Article Title: Comparative genomics and microbiome profiling reveal a conserved Vibrio rich mucus microbiota of the Florida false coral ( Ricordea florida )

doi: 10.64898/2026.09.17.752451

Figure Lengend Snippet: A. Relative abundance (%) of bacteria Genera collected from polyps from distinct tank systems reveals a dominance of Vibrio . Columns of each bar graph represent a polyp (A or B) and tank collected from is noted with the number. B. Polyps from the same tank show no difference in bacterial assemblage. A principal component analysis (PCA) reveals no differences in structure of bacterial communities between tank systems (12, 14, and 15 represented by blue, green and purple circles). X and Y axes show Principal Component 1 and Principal Component 2 that explain 56% and 22% of the total variance, respectively. (C). Shared and unique ASVs among corallimorph-associated bacterial communities. UpSet plot showing the overlap of amplicon sequence variants (ASVs) among Tanks 12, 14, and 15. An ASV was considered present within a tank only if it was detected in both animals sampled from that tank. Numbers indicate ASVs unique to individual tanks or shared among multiple tanks.

Article Snippet: Some isolates were re-sequenced using the Plasmidsaurus Whole Genome Sequencing service (Oxford Nanopore, R10.4.1 long-reads and de novo genome assembly).

Techniques: Bacteria, Analysis, Amplification, Sequencing

HeLa:Cas9 cells in 10cm plates were transfected with overexpression constructs for HPV16L2, encoding a C-terminal HA tag, and either an overexpression construct for PLCδ4 or PLCγ1, both containing the sequence for both C-terminal FLAG tag and c-Myc tag, or pCMV6, an empty vector control. Transfected cells were harvested 24h post-transfection, lysed in buffer containing 1% NP40 and 0.1% SDS, and then (cleared) lysates were incubated with magnetic beads coupled to anti-HA antibodies to pull down L2. Co-immunoprecipitation (CoIP) of PLCδ4 in bead elutions (“Elutes”) was determined using Western blot. Western blotting against the HA and c-Myc tags was used to confirm the presence of L2, PLCδ4, and PLCγ1 in “elutes” and “cleared lysates”. Blots are representative of three independent experiments ( N =3).

Journal: bioRxiv

Article Title: HPV16 Utilizes Phospholipase C(s) for its Genome Egress

doi: 10.64898/2026.09.03.749318

Figure Lengend Snippet: HeLa:Cas9 cells in 10cm plates were transfected with overexpression constructs for HPV16L2, encoding a C-terminal HA tag, and either an overexpression construct for PLCδ4 or PLCγ1, both containing the sequence for both C-terminal FLAG tag and c-Myc tag, or pCMV6, an empty vector control. Transfected cells were harvested 24h post-transfection, lysed in buffer containing 1% NP40 and 0.1% SDS, and then (cleared) lysates were incubated with magnetic beads coupled to anti-HA antibodies to pull down L2. Co-immunoprecipitation (CoIP) of PLCδ4 in bead elutions (“Elutes”) was determined using Western blot. Western blotting against the HA and c-Myc tags was used to confirm the presence of L2, PLCδ4, and PLCγ1 in “elutes” and “cleared lysates”. Blots are representative of three independent experiments ( N =3).

Article Snippet: PLCβ4 and PLCδ4 gene edits in selected clonal HeLa cells were further confirmed via sequencing with Plasmidsaurus sequencing services using Oxford Nanopore Technology.

Techniques: Transfection, Over Expression, Construct, Sequencing, FLAG-tag, Plasmid Preparation, Control, Incubation, Magnetic Beads, Co-Immunoprecipitation Assay, Western Blot

Step 1 ) The sensors are printed with biodegradable PHBV, poly(3-hydroxybutuyrate- co -3-hydroxyvalerate), on one side and non-biodegradable PMMA, poly(methyl methacrylate) on the other side as a control. Step 2 ) UV-sterilized sensors are inserted into soil mesocosms and monitor PHBV degradation in real-time as an increase in resistance, which we normalize to the non-degraded PMMA. Step 3 ) At the end of the mesocosm incubation, the sensors are removed and we can summarise the PHBV degradation signal to identify which soils have the highest capacity to degrade PHBV. Step 4 ) PHBV and PMMA material from sensors incubated in the highest PHBV-degrading soils are scraped off and used as starting material for culture-independent ( Step 5 , left panel) and culture-dependent ( Step 6 , right panel) approaches to identify novel PHBV degraders. Step 5, left ) We used the PHBV and PMMA material from high degrading soils for 16S rRNA and ITS marker gene sequencing to identify microbes enriched on degraded PHBV compared to non-degraded PMMA from each in situ sensor. Step 5, right ) We also used the PHBV material from high degrading soils to isolate novel PHBV degraders on agar plates with a PHBV overlay (inset). Isolates that formed clearing zones on plates were cultured in liquid media to measure their continued capacity to degrade PHBV. We also measured the respiration rates of isolates growing with PHBV as their sole carbon source to confirm PHBV catabolism.

Journal: bioRxiv

Article Title: Identification of soil microbes associated with real-time plastic degradation using in situ conductivity sensors

doi: 10.64898/2026.08.16.745074

Figure Lengend Snippet: Step 1 ) The sensors are printed with biodegradable PHBV, poly(3-hydroxybutuyrate- co -3-hydroxyvalerate), on one side and non-biodegradable PMMA, poly(methyl methacrylate) on the other side as a control. Step 2 ) UV-sterilized sensors are inserted into soil mesocosms and monitor PHBV degradation in real-time as an increase in resistance, which we normalize to the non-degraded PMMA. Step 3 ) At the end of the mesocosm incubation, the sensors are removed and we can summarise the PHBV degradation signal to identify which soils have the highest capacity to degrade PHBV. Step 4 ) PHBV and PMMA material from sensors incubated in the highest PHBV-degrading soils are scraped off and used as starting material for culture-independent ( Step 5 , left panel) and culture-dependent ( Step 6 , right panel) approaches to identify novel PHBV degraders. Step 5, left ) We used the PHBV and PMMA material from high degrading soils for 16S rRNA and ITS marker gene sequencing to identify microbes enriched on degraded PHBV compared to non-degraded PMMA from each in situ sensor. Step 5, right ) We also used the PHBV material from high degrading soils to isolate novel PHBV degraders on agar plates with a PHBV overlay (inset). Isolates that formed clearing zones on plates were cultured in liquid media to measure their continued capacity to degrade PHBV. We also measured the respiration rates of isolates growing with PHBV as their sole carbon source to confirm PHBV catabolism.

Article Snippet: DNA aliquots from two of our isolates, Streptomyces sp. [2CO Garden Isolate], Lysinibacillus sp. [2CO Garden Isolate] were sequenced by Plasmidsaurus using their bacterial genome sequencing service.

Techniques: Control, Incubation, Marker, Sequencing, In Situ, Cell Culture