Lentiviral vector packaging and producer cell lines yield titers equivalent to the industry-standard four-plasmid process.

Lentiviral vector (LVV)-mediated cell and gene therapies have the potential to cure diseases that currently require lifelong intervention. However, the requirement for plasmid transfection hinders large-scale LVV manufacture. Moreover, large-scale plasmid production, testing, and transfection contribute to operational risk and the high cost associated with this therapeutic modality.

Lambda Red Recombineering of Bacteriophage in the Lysogenic State.

We present a recombineering-based method for editing the genome of a temperate phage. The method uses the lambda Red recombination system to edit the genome of a lysogenized host with a prophage compatible with bacteriophage lambda. Linear DNA is used as the recombination substrate and antibiotic resistance is used as the basis for selection of recombinants.

Scarless Recombineering of Phage in Lysogenic State.

We present a scarless recombineering-based method for introducing multiple point mutations into the genome of a temperate phage. The method uses the λ Red recombineering system to promote exogenous ssDNA oligos to anneal on the prophage lagging strand during host genome replication. DNA repair is suppressed by inducing the expression of a dominant-negative mutant protein of the methyl-directed mismatch repair system.

Engineering Bacteria to Produce Pure Phage-like Particles for Gene Delivery.

Natural and engineered phages have been used in many applications, but their use to deliver user-defined genetic cargoes has been hampered by contamination with replicative phage, restricting use of the technology beyond the laboratory. Here we present a method to produce transducing particles without contamination. In addition, we demonstrate the use of a helper phage-free transducing particle preparation as an antimicrobial agent.

Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7.

With the recent rise in interest in using lytic bacteriophages as therapeutic agents, there is an urgent requirement to understand their fundamental biology to enable the engineering of their genomes. Current methods of phage engineering rely on homologous recombination, followed by a system of selection to identify recombinant phages. For bacteriophage T7, the host genes or have been used as a selection mechanism along with both type I and II CRISPR systems to select against wild-type phage and enrich for the desired mutant.

Nucleotide signaling pathway convergence in a cAMP-sensing bacterial c-di-GMP phosphodiesterase.

Bacterial usage of the cyclic dinucleotide c-di-GMP is widespread, governing the transition between motile/sessile and unicellular/multicellular behaviors. There is limited information on c-di-GMP metabolism, particularly on regulatory mechanisms governing control of EAL c-di-GMP phosphodiesterases. Herein, we provide high-resolution structures for an EAL enzyme Bd1971, from the predatory bacterium Bdellovibrio bacteriovorus, which is controlled by a second signaling nucleotide, cAMP.

Linear dichroism of visible-region chromophores using M13 bacteriophage as an alignment scaffold.

It is a challenge within the field of biomimetics to recreate the properties of light-harvesting antennae found in plants and photosynthetic bacteria. Attempts to recreate these biological structures typically rely on the alignment of fluorescent moieties attachment to an inert linear scaffold, DNA, RNA or amyloid fibrils, to enable Förster resonance energy transfer (FRET) between attached chromophores. While there has been some success in this approach, refinement of the alignment of the chromophores is often limited, which may limit the efficiency of energy transfer achieved.

Dye Aggregate-Mediated Self-Assembly of Bacteriophage Bioconjugates.

One of the central themes of biomolecular engineering is the challenge of exploiting the properties of biological materials. Part of this challenge has been uncovering and harnessing properties of biological components that only emerge following their ordered self-assembly. One biomolecular building block that has received significant interest in the past decade is the M13 bacteriophage.

Mutation of M13 Bacteriophage Major Coat Protein for Increased Conjugation to Exogenous Compounds.

Over the past ten years there has been increasing interest in the conjugation of exogenous compounds to the surface of the M13 bacteriophage. M13 offers a convenient scaffold for the development of nanoassemblies with useful functions, such as highly specific drug delivery and pathogen detection. However, the progress of these technologies has been hindered by the limited efficiency of conjugation to the bacteriophage.