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conventional non replicating mrna based therapeutics  (Moderna)

 
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    Structured Review

    Moderna conventional non replicating mrna based therapeutics
    Conventional Non Replicating Mrna Based Therapeutics, supplied by Moderna, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/replication/messenger+mrna+rna+vaccines/pmc13264025-10-0-14
    Average 86 stars, based on 1 article reviews
    conventional non replicating mrna based therapeutics - by Bioz Stars, 2026-10
    86/100 stars

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    Related Articles

    Vaccines:

    Article Title: Evidence-based assessment of safety and mechanistic questions Related to mRNA COVID-19 Vaccines.
    Article Snippet: .. Goody , David W. Reid , Darin K. Edwards , Veronica Urdaneta , Brian P. Doyle , Walter L. Straus , Carole Henry , Bishoy Rizkalla Moderna, Inc., Cambridge, MA, USA A R T I C L E I N F O Keywords: mRNA vaccines COVID-19 Vaccine safety Residual DNA Biodistribution, misinformation A B S T R A C T Messenger RNA (mRNA) COVID-19 vaccines have been administered globally at unprecedented scale and have demonstrated strong effectiveness against severe disease, hospitalization, and death. ..

    Article Title: Vaccination should not be forgotten in patients receiving immunoglobulin replacement therapy
    Article Snippet: .. P.L. serves as an adviser to the Adult Immunization Board (supported by an unrestricted grant from Vaccines Europe); has been a scientific expert on respiratory syncytial virus vaccines for GSK and on COVID-19, influenza, and cytomegalovirus vaccines for Moderna. ..

    Article Title: A Systemic Selective Modified mRNA Delivery Platform for Preventing Chemotherapy-Induced Cardiotoxicity.
    Article Snippet: .. Nucleoside-modified messenger RNA (modRNA) therapies are a novel class of drugs that gained widespread attention following the success of COVID-19 mRNA vaccines produced by Moderna and Pfizer-BioNTech [ 21 ]. ..

    Article Title: Long-term exposure to fine particle air pollution associated with emergency COVID-19 hospitalizations: A nationwide retrospective Korean cohort study.
    Article Snippet: Objectives: Emerging evidence suggests an association between ambient air pollution and severe coronavirus disease 2019 (COVID-19) outcomes.. We aimed to assess whether long-term exposure to fine particulate matter (PM2.5) is associated with acute hospitalization after COVID-19 infection and whether this association is modified by COVID-19 vaccination.. Study design: Retrospective nationwide cohort study of confirmed COVID-19 cases in the Republic of Korea, with municipality-level PM2.5 exposure assigned and time to emergency admission modeled using a Cox's proportional hazards model. Methods: Using national health insurance data in the Republic of Korea, we enrolled 556,767 individuals confirmed with COVID-19 infection from October 8, 2020, to December 31, 2021.

    Article Title: Thyroid Eye Disease Following SARS-CoV-2 Vaccination: Experience of a Case Series.
    Article Snippet: .. Currently, four major vaccine platforms are used worldwide: messenger Vaccines 2026, 14, 37 https://doi.org/10.3390/vaccines14010037 RNA (mRNA) vaccines (Pfizer–BioNTech, Moderna), adenoviral vector vaccines (AstraZeneca, Janssen), inactivated virus vaccines, and protein subunit vaccines. ..

    Virus:

    Article Title: Vaccination should not be forgotten in patients receiving immunoglobulin replacement therapy
    Article Snippet: .. P.L. serves as an adviser to the Adult Immunization Board (supported by an unrestricted grant from Vaccines Europe); has been a scientific expert on respiratory syncytial virus vaccines for GSK and on COVID-19, influenza, and cytomegalovirus vaccines for Moderna. ..

    Article Title: Thyroid Eye Disease Following SARS-CoV-2 Vaccination: Experience of a Case Series.
    Article Snippet: .. Currently, four major vaccine platforms are used worldwide: messenger Vaccines 2026, 14, 37 https://doi.org/10.3390/vaccines14010037 RNA (mRNA) vaccines (Pfizer–BioNTech, Moderna), adenoviral vector vaccines (AstraZeneca, Janssen), inactivated virus vaccines, and protein subunit vaccines. ..

    Produced:

    Article Title: A Systemic Selective Modified mRNA Delivery Platform for Preventing Chemotherapy-Induced Cardiotoxicity.
    Article Snippet: .. Nucleoside-modified messenger RNA (modRNA) therapies are a novel class of drugs that gained widespread attention following the success of COVID-19 mRNA vaccines produced by Moderna and Pfizer-BioNTech [ 21 ]. ..

    Food & Beverages:

    Article Title: Long-term exposure to fine particle air pollution associated with emergency COVID-19 hospitalizations: A nationwide retrospective Korean cohort study.
    Article Snippet: Objectives: Emerging evidence suggests an association between ambient air pollution and severe coronavirus disease 2019 (COVID-19) outcomes.. We aimed to assess whether long-term exposure to fine particulate matter (PM2.5) is associated with acute hospitalization after COVID-19 infection and whether this association is modified by COVID-19 vaccination.. Study design: Retrospective nationwide cohort study of confirmed COVID-19 cases in the Republic of Korea, with municipality-level PM2.5 exposure assigned and time to emergency admission modeled using a Cox's proportional hazards model. Methods: Using national health insurance data in the Republic of Korea, we enrolled 556,767 individuals confirmed with COVID-19 infection from October 8, 2020, to December 31, 2021.

    In Vitro:

    Article Title: A self-amplifying RNA vector based on rubella virus for mRNA therapeutics and vaccine applications
    Article Snippet: .. Conventional non-replicating mRNA-based therapeutics are in vitro -transcribed RNAs encoding viral proteins, such as Moderna or BioNTech-Pfizer COVID-19 vaccine or therapeutic proteins, such as antibodies or immune modulators for immunotherapy., , They may incorporate chemically modified nucleotides, sequence optimization, and different purification strategies to improve the efficiency of mRNA translation and RNA stability. ..

    Modification:

    Article Title: A self-amplifying RNA vector based on rubella virus for mRNA therapeutics and vaccine applications
    Article Snippet: .. Conventional non-replicating mRNA-based therapeutics are in vitro -transcribed RNAs encoding viral proteins, such as Moderna or BioNTech-Pfizer COVID-19 vaccine or therapeutic proteins, such as antibodies or immune modulators for immunotherapy., , They may incorporate chemically modified nucleotides, sequence optimization, and different purification strategies to improve the efficiency of mRNA translation and RNA stability. ..

    Sequencing:

    Article Title: A self-amplifying RNA vector based on rubella virus for mRNA therapeutics and vaccine applications
    Article Snippet: .. Conventional non-replicating mRNA-based therapeutics are in vitro -transcribed RNAs encoding viral proteins, such as Moderna or BioNTech-Pfizer COVID-19 vaccine or therapeutic proteins, such as antibodies or immune modulators for immunotherapy., , They may incorporate chemically modified nucleotides, sequence optimization, and different purification strategies to improve the efficiency of mRNA translation and RNA stability. ..

    Purification:

    Article Title: A self-amplifying RNA vector based on rubella virus for mRNA therapeutics and vaccine applications
    Article Snippet: .. Conventional non-replicating mRNA-based therapeutics are in vitro -transcribed RNAs encoding viral proteins, such as Moderna or BioNTech-Pfizer COVID-19 vaccine or therapeutic proteins, such as antibodies or immune modulators for immunotherapy., , They may incorporate chemically modified nucleotides, sequence optimization, and different purification strategies to improve the efficiency of mRNA translation and RNA stability. ..

    Stability:

    Article Title: A self-amplifying RNA vector based on rubella virus for mRNA therapeutics and vaccine applications
    Article Snippet: .. Conventional non-replicating mRNA-based therapeutics are in vitro -transcribed RNAs encoding viral proteins, such as Moderna or BioNTech-Pfizer COVID-19 vaccine or therapeutic proteins, such as antibodies or immune modulators for immunotherapy., , They may incorporate chemically modified nucleotides, sequence optimization, and different purification strategies to improve the efficiency of mRNA translation and RNA stability. ..



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    (a) The PRD1 bacteriophage possesses a 14927 bp genome encoding four proteins involved in protein-primed DNA <t>replication:</t> the DNA polymerase P1, the terminal protein P8, and the single-stranded DNA-binding proteins P12 and P19. (b) PRIME comprises four plasmids: pORM ( O rthogonal R eplication M achinery), pOTH ( O rthogonal T ransformation H elper), pORFITR ( O rthogonal R eplicons F lanked by Inverted T erminal R epeats), and lopEG ( L inear O rthogonal P lasmid with E volving G enes). The pORM plasmid carries the genes required for protein-primed DNA replication under control of the inducible P Cin promoter. P12_T2 and P19 are indicated in brackets, as these proteins are dispensable for <t>orthogonal</t> plasmid replication but are present in the pORM_P12_T2 construct. pORM additionally contains the broad-host-range pBBR1 origin of replication, enabling propagation in a wide range of bacterial hosts. The pOTH plasmid encodes accessory proteins facilitating lopEG transformation. Owing to their toxicity, these proteins are expressed under control of the inducible P BAD promoter. The temperature-sensitive pSC101(ts) origin enables efficient curing of pOTH following lopEG establishment. The pORFITR plasmid facilitates cloning and production of lopEG plasmids. It contains two inverted terminal repeat (ITR) regions flanked by MlyI recognition sites. As a Type IIS restriction enzyme, MlyI cleaves outside of its recognition sequence, enabling blunt cleavage precisely at the edges of the ITRs. The R6K conditional origin of replication prevents plasmid propagation in cells lacking the Pir protein, allowing direct transformation of lopEG plasmids without gel purification. (c) Workflow for establishment of orthogonal plasmids in E. coli . (1) Cells are first transformed with pORM and pOTH. (2) Expression of the replication proteins is induced, and the temperature is increased prior to lopEG transformation to inhibit further replication of pOTH. (3) lopEG plasmids are electroporated into competent cells expressing the PRD1 replication machinery. Orthogonal plasmid copy number can subsequently be tuned using the OHC14-HSL inducer. (d) PRIME enables continuous directed evolution experiments. Replacement of the wild-type P1 polymerase (green) with an error-prone variant (red) promotes the generation of DNA libraries directly in vivo without the need for repeated manual intervention. Iterative cycles of diversification and selection subsequently enable the rapid evolution of constructs with improved properties.
    Orthogonal Replication Machinery, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    replicate analyses  (Agilent technologies)
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    (a) The PRD1 bacteriophage possesses a 14927 bp genome encoding four proteins involved in protein-primed DNA <t>replication:</t> the DNA polymerase P1, the terminal protein P8, and the single-stranded DNA-binding proteins P12 and P19. (b) PRIME comprises four plasmids: pORM ( O rthogonal R eplication M achinery), pOTH ( O rthogonal T ransformation H elper), pORFITR ( O rthogonal R eplicons F lanked by Inverted T erminal R epeats), and lopEG ( L inear O rthogonal P lasmid with E volving G enes). The pORM plasmid carries the genes required for protein-primed DNA replication under control of the inducible P Cin promoter. P12_T2 and P19 are indicated in brackets, as these proteins are dispensable for <t>orthogonal</t> plasmid replication but are present in the pORM_P12_T2 construct. pORM additionally contains the broad-host-range pBBR1 origin of replication, enabling propagation in a wide range of bacterial hosts. The pOTH plasmid encodes accessory proteins facilitating lopEG transformation. Owing to their toxicity, these proteins are expressed under control of the inducible P BAD promoter. The temperature-sensitive pSC101(ts) origin enables efficient curing of pOTH following lopEG establishment. The pORFITR plasmid facilitates cloning and production of lopEG plasmids. It contains two inverted terminal repeat (ITR) regions flanked by MlyI recognition sites. As a Type IIS restriction enzyme, MlyI cleaves outside of its recognition sequence, enabling blunt cleavage precisely at the edges of the ITRs. The R6K conditional origin of replication prevents plasmid propagation in cells lacking the Pir protein, allowing direct transformation of lopEG plasmids without gel purification. (c) Workflow for establishment of orthogonal plasmids in E. coli . (1) Cells are first transformed with pORM and pOTH. (2) Expression of the replication proteins is induced, and the temperature is increased prior to lopEG transformation to inhibit further replication of pOTH. (3) lopEG plasmids are electroporated into competent cells expressing the PRD1 replication machinery. Orthogonal plasmid copy number can subsequently be tuned using the OHC14-HSL inducer. (d) PRIME enables continuous directed evolution experiments. Replacement of the wild-type P1 polymerase (green) with an error-prone variant (red) promotes the generation of DNA libraries directly in vivo without the need for repeated manual intervention. Iterative cycles of diversification and selection subsequently enable the rapid evolution of constructs with improved properties.
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    (a) The PRD1 bacteriophage possesses a 14927 bp genome encoding four proteins involved in protein-primed DNA <t>replication:</t> the DNA polymerase P1, the terminal protein P8, and the single-stranded DNA-binding proteins P12 and P19. (b) PRIME comprises four plasmids: pORM ( O rthogonal R eplication M achinery), pOTH ( O rthogonal T ransformation H elper), pORFITR ( O rthogonal R eplicons F lanked by Inverted T erminal R epeats), and lopEG ( L inear O rthogonal P lasmid with E volving G enes). The pORM plasmid carries the genes required for protein-primed DNA replication under control of the inducible P Cin promoter. P12_T2 and P19 are indicated in brackets, as these proteins are dispensable for <t>orthogonal</t> plasmid replication but are present in the pORM_P12_T2 construct. pORM additionally contains the broad-host-range pBBR1 origin of replication, enabling propagation in a wide range of bacterial hosts. The pOTH plasmid encodes accessory proteins facilitating lopEG transformation. Owing to their toxicity, these proteins are expressed under control of the inducible P BAD promoter. The temperature-sensitive pSC101(ts) origin enables efficient curing of pOTH following lopEG establishment. The pORFITR plasmid facilitates cloning and production of lopEG plasmids. It contains two inverted terminal repeat (ITR) regions flanked by MlyI recognition sites. As a Type IIS restriction enzyme, MlyI cleaves outside of its recognition sequence, enabling blunt cleavage precisely at the edges of the ITRs. The R6K conditional origin of replication prevents plasmid propagation in cells lacking the Pir protein, allowing direct transformation of lopEG plasmids without gel purification. (c) Workflow for establishment of orthogonal plasmids in E. coli . (1) Cells are first transformed with pORM and pOTH. (2) Expression of the replication proteins is induced, and the temperature is increased prior to lopEG transformation to inhibit further replication of pOTH. (3) lopEG plasmids are electroporated into competent cells expressing the PRD1 replication machinery. Orthogonal plasmid copy number can subsequently be tuned using the OHC14-HSL inducer. (d) PRIME enables continuous directed evolution experiments. Replacement of the wild-type P1 polymerase (green) with an error-prone variant (red) promotes the generation of DNA libraries directly in vivo without the need for repeated manual intervention. Iterative cycles of diversification and selection subsequently enable the rapid evolution of constructs with improved properties.
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    96 pin replicator  (Boekel Scientific)
    86
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    (a) The PRD1 bacteriophage possesses a 14927 bp genome encoding four proteins involved in protein-primed DNA <t>replication:</t> the DNA polymerase P1, the terminal protein P8, and the single-stranded DNA-binding proteins P12 and P19. (b) PRIME comprises four plasmids: pORM ( O rthogonal R eplication M achinery), pOTH ( O rthogonal T ransformation H elper), pORFITR ( O rthogonal R eplicons F lanked by Inverted T erminal R epeats), and lopEG ( L inear O rthogonal P lasmid with E volving G enes). The pORM plasmid carries the genes required for protein-primed DNA replication under control of the inducible P Cin promoter. P12_T2 and P19 are indicated in brackets, as these proteins are dispensable for <t>orthogonal</t> plasmid replication but are present in the pORM_P12_T2 construct. pORM additionally contains the broad-host-range pBBR1 origin of replication, enabling propagation in a wide range of bacterial hosts. The pOTH plasmid encodes accessory proteins facilitating lopEG transformation. Owing to their toxicity, these proteins are expressed under control of the inducible P BAD promoter. The temperature-sensitive pSC101(ts) origin enables efficient curing of pOTH following lopEG establishment. The pORFITR plasmid facilitates cloning and production of lopEG plasmids. It contains two inverted terminal repeat (ITR) regions flanked by MlyI recognition sites. As a Type IIS restriction enzyme, MlyI cleaves outside of its recognition sequence, enabling blunt cleavage precisely at the edges of the ITRs. The R6K conditional origin of replication prevents plasmid propagation in cells lacking the Pir protein, allowing direct transformation of lopEG plasmids without gel purification. (c) Workflow for establishment of orthogonal plasmids in E. coli . (1) Cells are first transformed with pORM and pOTH. (2) Expression of the replication proteins is induced, and the temperature is increased prior to lopEG transformation to inhibit further replication of pOTH. (3) lopEG plasmids are electroporated into competent cells expressing the PRD1 replication machinery. Orthogonal plasmid copy number can subsequently be tuned using the OHC14-HSL inducer. (d) PRIME enables continuous directed evolution experiments. Replacement of the wild-type P1 polymerase (green) with an error-prone variant (red) promotes the generation of DNA libraries directly in vivo without the need for repeated manual intervention. Iterative cycles of diversification and selection subsequently enable the rapid evolution of constructs with improved properties.
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    replication  (Bavarian Nordic)
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    (a) The PRD1 bacteriophage possesses a 14927 bp genome encoding four proteins involved in protein-primed DNA <t>replication:</t> the DNA polymerase P1, the terminal protein P8, and the single-stranded DNA-binding proteins P12 and P19. (b) PRIME comprises four plasmids: pORM ( O rthogonal R eplication M achinery), pOTH ( O rthogonal T ransformation H elper), pORFITR ( O rthogonal R eplicons F lanked by Inverted T erminal R epeats), and lopEG ( L inear O rthogonal P lasmid with E volving G enes). The pORM plasmid carries the genes required for protein-primed DNA replication under control of the inducible P Cin promoter. P12_T2 and P19 are indicated in brackets, as these proteins are dispensable for <t>orthogonal</t> plasmid replication but are present in the pORM_P12_T2 construct. pORM additionally contains the broad-host-range pBBR1 origin of replication, enabling propagation in a wide range of bacterial hosts. The pOTH plasmid encodes accessory proteins facilitating lopEG transformation. Owing to their toxicity, these proteins are expressed under control of the inducible P BAD promoter. The temperature-sensitive pSC101(ts) origin enables efficient curing of pOTH following lopEG establishment. The pORFITR plasmid facilitates cloning and production of lopEG plasmids. It contains two inverted terminal repeat (ITR) regions flanked by MlyI recognition sites. As a Type IIS restriction enzyme, MlyI cleaves outside of its recognition sequence, enabling blunt cleavage precisely at the edges of the ITRs. The R6K conditional origin of replication prevents plasmid propagation in cells lacking the Pir protein, allowing direct transformation of lopEG plasmids without gel purification. (c) Workflow for establishment of orthogonal plasmids in E. coli . (1) Cells are first transformed with pORM and pOTH. (2) Expression of the replication proteins is induced, and the temperature is increased prior to lopEG transformation to inhibit further replication of pOTH. (3) lopEG plasmids are electroporated into competent cells expressing the PRD1 replication machinery. Orthogonal plasmid copy number can subsequently be tuned using the OHC14-HSL inducer. (d) PRIME enables continuous directed evolution experiments. Replacement of the wild-type P1 polymerase (green) with an error-prone variant (red) promotes the generation of DNA libraries directly in vivo without the need for repeated manual intervention. Iterative cycles of diversification and selection subsequently enable the rapid evolution of constructs with improved properties.
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    94
    MedChemExpress replication
    (a) The PRD1 bacteriophage possesses a 14927 bp genome encoding four proteins involved in protein-primed DNA <t>replication:</t> the DNA polymerase P1, the terminal protein P8, and the single-stranded DNA-binding proteins P12 and P19. (b) PRIME comprises four plasmids: pORM ( O rthogonal R eplication M achinery), pOTH ( O rthogonal T ransformation H elper), pORFITR ( O rthogonal R eplicons F lanked by Inverted T erminal R epeats), and lopEG ( L inear O rthogonal P lasmid with E volving G enes). The pORM plasmid carries the genes required for protein-primed DNA replication under control of the inducible P Cin promoter. P12_T2 and P19 are indicated in brackets, as these proteins are dispensable for <t>orthogonal</t> plasmid replication but are present in the pORM_P12_T2 construct. pORM additionally contains the broad-host-range pBBR1 origin of replication, enabling propagation in a wide range of bacterial hosts. The pOTH plasmid encodes accessory proteins facilitating lopEG transformation. Owing to their toxicity, these proteins are expressed under control of the inducible P BAD promoter. The temperature-sensitive pSC101(ts) origin enables efficient curing of pOTH following lopEG establishment. The pORFITR plasmid facilitates cloning and production of lopEG plasmids. It contains two inverted terminal repeat (ITR) regions flanked by MlyI recognition sites. As a Type IIS restriction enzyme, MlyI cleaves outside of its recognition sequence, enabling blunt cleavage precisely at the edges of the ITRs. The R6K conditional origin of replication prevents plasmid propagation in cells lacking the Pir protein, allowing direct transformation of lopEG plasmids without gel purification. (c) Workflow for establishment of orthogonal plasmids in E. coli . (1) Cells are first transformed with pORM and pOTH. (2) Expression of the replication proteins is induced, and the temperature is increased prior to lopEG transformation to inhibit further replication of pOTH. (3) lopEG plasmids are electroporated into competent cells expressing the PRD1 replication machinery. Orthogonal plasmid copy number can subsequently be tuned using the OHC14-HSL inducer. (d) PRIME enables continuous directed evolution experiments. Replacement of the wild-type P1 polymerase (green) with an error-prone variant (red) promotes the generation of DNA libraries directly in vivo without the need for repeated manual intervention. Iterative cycles of diversification and selection subsequently enable the rapid evolution of constructs with improved properties.
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    pcr replicates  (Taxon Biosciences)
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    Taxon Biosciences pcr replicates
    (a) The PRD1 bacteriophage possesses a 14927 bp genome encoding four proteins involved in protein-primed DNA <t>replication:</t> the DNA polymerase P1, the terminal protein P8, and the single-stranded DNA-binding proteins P12 and P19. (b) PRIME comprises four plasmids: pORM ( O rthogonal R eplication M achinery), pOTH ( O rthogonal T ransformation H elper), pORFITR ( O rthogonal R eplicons F lanked by Inverted T erminal R epeats), and lopEG ( L inear O rthogonal P lasmid with E volving G enes). The pORM plasmid carries the genes required for protein-primed DNA replication under control of the inducible P Cin promoter. P12_T2 and P19 are indicated in brackets, as these proteins are dispensable for <t>orthogonal</t> plasmid replication but are present in the pORM_P12_T2 construct. pORM additionally contains the broad-host-range pBBR1 origin of replication, enabling propagation in a wide range of bacterial hosts. The pOTH plasmid encodes accessory proteins facilitating lopEG transformation. Owing to their toxicity, these proteins are expressed under control of the inducible P BAD promoter. The temperature-sensitive pSC101(ts) origin enables efficient curing of pOTH following lopEG establishment. The pORFITR plasmid facilitates cloning and production of lopEG plasmids. It contains two inverted terminal repeat (ITR) regions flanked by MlyI recognition sites. As a Type IIS restriction enzyme, MlyI cleaves outside of its recognition sequence, enabling blunt cleavage precisely at the edges of the ITRs. The R6K conditional origin of replication prevents plasmid propagation in cells lacking the Pir protein, allowing direct transformation of lopEG plasmids without gel purification. (c) Workflow for establishment of orthogonal plasmids in E. coli . (1) Cells are first transformed with pORM and pOTH. (2) Expression of the replication proteins is induced, and the temperature is increased prior to lopEG transformation to inhibit further replication of pOTH. (3) lopEG plasmids are electroporated into competent cells expressing the PRD1 replication machinery. Orthogonal plasmid copy number can subsequently be tuned using the OHC14-HSL inducer. (d) PRIME enables continuous directed evolution experiments. Replacement of the wild-type P1 polymerase (green) with an error-prone variant (red) promotes the generation of DNA libraries directly in vivo without the need for repeated manual intervention. Iterative cycles of diversification and selection subsequently enable the rapid evolution of constructs with improved properties.
    Pcr Replicates, supplied by Taxon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (a) The PRD1 bacteriophage possesses a 14927 bp genome encoding four proteins involved in protein-primed DNA replication: the DNA polymerase P1, the terminal protein P8, and the single-stranded DNA-binding proteins P12 and P19. (b) PRIME comprises four plasmids: pORM ( O rthogonal R eplication M achinery), pOTH ( O rthogonal T ransformation H elper), pORFITR ( O rthogonal R eplicons F lanked by Inverted T erminal R epeats), and lopEG ( L inear O rthogonal P lasmid with E volving G enes). The pORM plasmid carries the genes required for protein-primed DNA replication under control of the inducible P Cin promoter. P12_T2 and P19 are indicated in brackets, as these proteins are dispensable for orthogonal plasmid replication but are present in the pORM_P12_T2 construct. pORM additionally contains the broad-host-range pBBR1 origin of replication, enabling propagation in a wide range of bacterial hosts. The pOTH plasmid encodes accessory proteins facilitating lopEG transformation. Owing to their toxicity, these proteins are expressed under control of the inducible P BAD promoter. The temperature-sensitive pSC101(ts) origin enables efficient curing of pOTH following lopEG establishment. The pORFITR plasmid facilitates cloning and production of lopEG plasmids. It contains two inverted terminal repeat (ITR) regions flanked by MlyI recognition sites. As a Type IIS restriction enzyme, MlyI cleaves outside of its recognition sequence, enabling blunt cleavage precisely at the edges of the ITRs. The R6K conditional origin of replication prevents plasmid propagation in cells lacking the Pir protein, allowing direct transformation of lopEG plasmids without gel purification. (c) Workflow for establishment of orthogonal plasmids in E. coli . (1) Cells are first transformed with pORM and pOTH. (2) Expression of the replication proteins is induced, and the temperature is increased prior to lopEG transformation to inhibit further replication of pOTH. (3) lopEG plasmids are electroporated into competent cells expressing the PRD1 replication machinery. Orthogonal plasmid copy number can subsequently be tuned using the OHC14-HSL inducer. (d) PRIME enables continuous directed evolution experiments. Replacement of the wild-type P1 polymerase (green) with an error-prone variant (red) promotes the generation of DNA libraries directly in vivo without the need for repeated manual intervention. Iterative cycles of diversification and selection subsequently enable the rapid evolution of constructs with improved properties.

    Journal: bioRxiv

    Article Title: PRIME: A modular plasmid-based platform for continuous in vivo evolution across bacteria

    doi: 10.64898/2026.09.28.754915

    Figure Lengend Snippet: (a) The PRD1 bacteriophage possesses a 14927 bp genome encoding four proteins involved in protein-primed DNA replication: the DNA polymerase P1, the terminal protein P8, and the single-stranded DNA-binding proteins P12 and P19. (b) PRIME comprises four plasmids: pORM ( O rthogonal R eplication M achinery), pOTH ( O rthogonal T ransformation H elper), pORFITR ( O rthogonal R eplicons F lanked by Inverted T erminal R epeats), and lopEG ( L inear O rthogonal P lasmid with E volving G enes). The pORM plasmid carries the genes required for protein-primed DNA replication under control of the inducible P Cin promoter. P12_T2 and P19 are indicated in brackets, as these proteins are dispensable for orthogonal plasmid replication but are present in the pORM_P12_T2 construct. pORM additionally contains the broad-host-range pBBR1 origin of replication, enabling propagation in a wide range of bacterial hosts. The pOTH plasmid encodes accessory proteins facilitating lopEG transformation. Owing to their toxicity, these proteins are expressed under control of the inducible P BAD promoter. The temperature-sensitive pSC101(ts) origin enables efficient curing of pOTH following lopEG establishment. The pORFITR plasmid facilitates cloning and production of lopEG plasmids. It contains two inverted terminal repeat (ITR) regions flanked by MlyI recognition sites. As a Type IIS restriction enzyme, MlyI cleaves outside of its recognition sequence, enabling blunt cleavage precisely at the edges of the ITRs. The R6K conditional origin of replication prevents plasmid propagation in cells lacking the Pir protein, allowing direct transformation of lopEG plasmids without gel purification. (c) Workflow for establishment of orthogonal plasmids in E. coli . (1) Cells are first transformed with pORM and pOTH. (2) Expression of the replication proteins is induced, and the temperature is increased prior to lopEG transformation to inhibit further replication of pOTH. (3) lopEG plasmids are electroporated into competent cells expressing the PRD1 replication machinery. Orthogonal plasmid copy number can subsequently be tuned using the OHC14-HSL inducer. (d) PRIME enables continuous directed evolution experiments. Replacement of the wild-type P1 polymerase (green) with an error-prone variant (red) promotes the generation of DNA libraries directly in vivo without the need for repeated manual intervention. Iterative cycles of diversification and selection subsequently enable the rapid evolution of constructs with improved properties.

    Article Snippet: The YFP gene was then replaced with the operon encoding for the orthogonal replication machinery by digestion with BsaI-HF-v2 and KpnI-HF restriction enzymes (New England Biolabs) and ligation with a similarly digested insert, yielding pORM_P12_P19.

    Techniques: DNA Binding Assay, Plasmid Preparation, Control, Construct, Transformation Assay, Toxicity, Cloning, Sequencing, Gel Purification, Expressing, Variant Assay, In Vivo, Selection, Protein Binding