A novel approach for T7 bacteriophage genome integration of exogenous DNA

Abstract

Background The comparatively small genome, well elucidated functional genomics and rapid life cycle confer T7 bacteriophage with great advantages for bio-application. Genetic manipulation of T7 genome plays a key role in T7 related applications. As one of the important aspects in T7 phage genetic modification, gene knock-in refers to two main approaches including direct genetic manipulation in vitro and recombineering. Neither of these available methods are efficient enough to support the development of innovative applications capitalizing on T7 bio-system and thus there is room for novel strategies that address this issue. Integration mediated by the ΦC31 integrase is one of the most robust site-specific recombination systems. ΦC31 integrases with enhanced activity and specificity have been developed such that it is ideal to effectuate exogenous DNA knock-in of T7 phage with advanced ΦC31 integrase. Methods Plasmid construction was conducted by routine molecular cloning technology. The engineered T7 bacteriophages were constructed through homologous recombination with corresponding plasmids and the functional T7 phage was designated as T7∆G10G11-attB. In the integration reaction, hosts with both executive plasmids (pEXM4) and donor plasmids (pMCBK) were lysed by T7∆G10G11-attB. Progenies of T7 phages that integrated with pMCBK were isolated in restrict hosts and validated by sequencing. T7∆G10G11-attB capacity limit was explored by another integration reactions with donor plasmids that contain exogenous DNA of various lengths. Results T7∆G10G11-attB exhibits abortive growth in restrictive hosts, and a bacterial attachment site recognized by ΦC31 integrase (attB) was confirmed to be present in the T7∆G10G11-attB genome via sequencing. The integration reaction demonstrated that plasmids containing the corresponding phage attachment site (attP) could be integrated into the T7∆G10G11-attB genome. The candidate recombinant phage was isolated and validated to have integrated exogenous DNA. The maximum capacity of T7∆G10G11-attB was explored, and it’s found that insertion of exogenous DNA sequences longer than 2 kbp long can be accommodated stably. Conclusion We advanced and established a novel approach for gene knock-in into the T7 genome using ΦC31 integrase.

PHEIGES, all-cell-free phage synthesis and selection from engineered genomes

“…PHEIGES provides a rapid, technically accessible, and low-cost method for phage engineering. Its DNA assembly efficiency (~10 10 PFU/ml) surpasses in vivo existing methods 15 15 17 18 and other cell-free phage engineering reports 20 39 based on Gibson assembly and additional purification, resulting in low phage cell-free synthesis (< 10 5 PFU/ml). PHEIGES efficiency is mirrored by the ease of engineering deletions and gene insertions at any permissive T7 loci into the T7 genome, serving as proof of concept for the versatility and accessibility of this workflow.…”

“…PHEIGES provides a rapid, technically accessible, and low-cost method for phage engineering. Its DNA assembly efficiency (∼10 10 PFU/ml) offers many advantages compared to the current in vivo methods 15 15 18 19 and other cell-free phage engineering methods 21 40 63 . The yeast cloning approach to phage genome engineering takes on the order of one week to achieve 64 65 .…”

“…Bacteriophages (phages) comprise an immense reservoir of biotechnologically-relevant bioactive materials such as DNA engineering tools 1 and phage display 2 with broad applications in phage therapy 3 4 5 6 7 8 , nanotechnology 9 10 , and vaccine scaffolds engineering 11 12 . Despite this, current in vivo phage engineering approaches, based on CRISPR 13 14 15 , yeast assembly 16 17 or integrases 18 limit phage’s usage due to time consuming cloning steps and complex selection processes 19 , especially challenging for obligated lytic phages 20 . On the other hand, in vitro genome engineering is emerging as a complementary approach to assemble and edit bacteriophages with novel properties.…”

Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield

“…T7 titers were determined using the standard double-layer agar (DLA) plaque assay [or DLA spot test] using BL21 as the host strain . Overnight cultures of BL21 in φLB were diluted 1:10 in prewarmed φLB and then incubated for 1 h to bring cultures to log-phase.…”

T7 Phage as an Emerging Nanobiomaterial with Genetically Tunable Target Specificity

“…[ 91 , 92 ] In addition, Liu et al. have developed ΦC31 integrase with enhanced activity and specificity of foreign gene knock‐in, [ 93 ] representing a new way for integrating foreign DNA into the T7 phage genome.…”