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Related Concept Videos

DNA Bacteriophages01:26

DNA Bacteriophages

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Bacteriophages, or phages, are viruses that specifically infect bacteria, utilizing their genetic material to hijack host cellular machinery for replication. DNA bacteriophages employ single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) genomes. These phages exhibit diverse replication strategies and host interactions, influencing their ecological roles and applications in biotechnology and medicine.ssDNA BacteriophagesssDNA phages, with their small genomes, utilize unique strategies to...
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Bacteriophages, also known as phages, are specialized viruses that infect bacteria. A key characteristic of phages is their distinctive “head-tail” morphology. A phage begins the infection process (i.e., lytic cycle) by attaching to the outside of a bacterial cell. Attachment is accomplished via proteins in the phage tail that bind to specific receptor proteins on the outer surface of the bacterium. The tail injects the phage’s DNA genome into the bacterial cytoplasm. In the...
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The lysogenic cycle is a crucial viral replication strategy that allows bacteriophages to persist within host cells without immediately destroying them. This process is primarily observed in temperate phages, such as bacteriophage lambda (λ), which infects Escherichia coli. The cycle allows the viral genome to persist across bacterial generations while keeping host cells viable.Integration of the Viral GenomeUpon infection, bacteriophage lambda attaches to the bacterial surface and injects...
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In contrast to the lytic cycle, phages infecting bacteria via the lysogenic cycle do not immediately kill their host cell. Instead, they combine their genome with the host genome, allowing the bacteria to replicate the phage DNA along with the bacterial genome. The incorporated copy of the phage genome is called the prophage. Some prophages can re-activate and enter the lytic cycle. This often occurs in response to a perturbation, such as DNA damage, but can also transpire in the absence of...
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Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System
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Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield.

Rani Brooks1, Lisa Morici2, Nicholas Sandoval3

  • 1Interdisciplinary Bioinnovation PhD Program, Tulane University, New Orleans, Louisiana 70118-5665, United States.

ACS Synthetic Biology
|August 7, 2023
PubMed
Summary

Cell-free bacteriophage synthesis (CFBS) enhances phage T7 production using engineered bacterial lysates. Modifying gene expression in transcription/translation machinery (TXTL) improved phage yields up to 10-fold, accelerating phage therapy development.

Keywords:
CRISRPiT7TX-TLcell-free bacteriophage synthesis (CFBS)cell-free expression systemsgene expression

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Infectious Disease Research

Background:

  • Phage therapy offers a promising alternative to antibiotics for drug-resistant infections but faces production challenges.
  • Cell-free bacteriophage synthesis (CFBS) is an in vitro method to produce phage virions, overcoming limitations of traditional methods.
  • Current CFBS systems using standard bacterial lysates are not optimized for high-yield bacteriophage production.

Purpose of the Study:

  • To enhance phage T7 yields in vitro using cell-free bacteriophage synthesis (CFBS).
  • To identify and manipulate specific bacterial genes to optimize transcription/translation machinery (TXTL) for improved phage production.
  • To demonstrate the potential of genetically modified bacterial strains for accelerating phage manufacturing.

Main Methods:

  • Utilized inducible CRISPR interference (CRISPRi) with nuclease-deficient Cas12a (dFnCas12a) to modulate the expression of 18 E. coli BL21 genes.
  • Overexpressed positive effector genes and repressed negative effector genes to modify the TXTL genetic background.
  • Quantified phage T7 yields produced via CFBS using the optimized TXTL.

Main Results:

  • Engineered E. coli BL21 TXTL significantly enhanced phage T7 CFBS yields by up to 10-fold in vitro.
  • Overexpression of translation initiation factor IF-3 (infC), and small RNAs OxyS and CyaR, boosted phage yields.
  • Repression of the RecC subunit of RecBCD exonuclease also contributed to increased phage production.

Conclusions:

  • Optimizing bacterial TXTL through genetic engineering is a viable strategy to overcome phage manufacturing bottlenecks.
  • Enhanced CFBS holds significant potential for accelerating the development and widespread adoption of phage therapy.
  • This study provides a foundation for further improvements in cell-free phage production systems.