Related Experiment Video

Updated: Jan 11, 2026

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System
11:33

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

Published on: August 17, 2017

15.4K

Genome synthesis, assembly, and rebooting of therapeutically useful high G+C% mycobacteriophages

Ching-Chung Ko1, Andrew P Sikkema2, Michael J Lauer1

  • 1Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260.

Proceedings of the National Academy of Sciences of the United States of America
|November 10, 2025
PubMed

Insights

Synthetic biology enables the creation of novel bacteriophages for treating nontuberculous mycobacteria (NTM). This research developed methods to synthesize and modify mycobacteriophage genomes, expanding therapeutic options.

Area of Science:

  • Synthetic biology
  • Virology
  • Genetics

Background:

  • Bacteriophages are promising therapeutics for bacterial infections, including nontuberculous mycobacteria (NTM).
  • Limited availability and variable infection profiles of natural phages hinder therapeutic development.
  • Mycobacteriophage genomes are large and have high G+C content, posing challenges for synthetic manipulation.

Purpose of the Study:

  • To develop efficient methods for de novo synthesis and genetic engineering of mycobacteriophage genomes.
  • To overcome challenges associated with high G+C content and large genome size in mycobacteriophages.
  • To expand the repertoire of mycobacteriophages for therapeutic applications and mycobacterial research.

Main Methods:

  • Utilized terminal deoxynucleotidyl transferase chemistry for high G+C% DNA synthesis.
  • Employed High-Complexity Golden Gate Assembly for complete mycobacteriophage genome reconstruction.
  • Demonstrated efficient genome rebooting via electroporation into *Mycobacterium smegmatis*.

Main Results:

  • Successfully synthesized genomes of phages BPs (41.9 kbp, 66.6% G+C%) and Bxb1 (50.5 kbp, 63.6% G+C%).
  • Constructed synthetic phage variants with targeted mutations and added genetic payloads.
  • Validated the efficiency of the synthetic genome assembly and rebooting process.

Conclusions:

  • Synthetic construction provides a versatile platform for creating custom mycobacteriophages.
  • This approach expands the toolkit for phage-based therapeutics against NTM.
  • Enables advancements in mycobacterial genetics and phage-based clinical applications.
Keywords:
bacteriophagesphage therapysynthetic genomics

More Related Videos

The MultiBac Protein Complex Production Platform at the EMBL13:51

The MultiBac Protein Complex Production Platform at the EMBL

Published on: July 11, 2013

16.5K
A Customizable Protocol for String Assembly gRNA Cloning STAgR10:00

A Customizable Protocol for String Assembly gRNA Cloning STAgR

Published on: December 26, 2018

10.1K

Related Concept Videos

DNA Bacteriophages01:26

DNA Bacteriophages

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...
773
Lysogenic Cycle of Bacteriophages00:43

Lysogenic Cycle of Bacteriophages

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...
67.3K
Lytic Cycle of Bacteriophages01:30

Lytic Cycle of Bacteriophages

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...
77.3K
Homologous Recombination02:31

Homologous Recombination

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
62.5K
Viral Recombination00:57

Viral Recombination

Cells are sometimes infected by more than one virus at once. When two viruses disassemble to expose their genomes for replication in the same cell, similar regions of their genomes can pair together and exchange sequences in a process called recombination. Alternatively, viruses with segmented genomes can swap segments in a process called reassortment.
24.9K
Synthetic Biology02:55

Synthetic Biology

Synthetic biology is an interdisciplinary science that involves using principles from disciplines such as engineering, molecular biology, cell biology, and systems biology. It involves remodeling existing organisms from nature or constructing completely new synthetic organisms for applications such as protein or enzyme production, bioremediation, value-added macromolecule production, and the addition of desirable traits to crops, to name a few.
Golden rice
Golden rice is a genetically modified...
5.5K