Rapid and Accurate Assembly of Large DNA Assisted by In Vitro Packaging of Bacteriophage

Abstract

Development of DNA assembly methods made it possible to construct large DNA. However, achieving a large DNA assembly easily, accurately, and at a low cost remains a challenge. This study shows that DNA assembled only by annealing of overlapping single-stranded DNA ends, which are generated by exonuclease treatment, without ligation can be packaged in phage particles and can also be transduced into bacterial cells. Based on this, I developed a simple method to construct long DNA of about 40–50 kb from five to ten PCR fragments using the bacteriophage in vitro packaging system. This method, namely, iPac ( in vitro Packaging-assisted DNA assembly), allowed accurate and rapid construction of large plasmids and phage genomes. This simple method will accelerate research in molecular and synthetic biology, including the construction of gene circuits or the engineering of metabolic pathways.

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

“…Other methods, such as SLiCE 43 , also do not interface well with TXTL as the ligation reaction inhibits cell-free gene expression (data not shown). Our attention focused on a recent method to assemble and package phage genomes into procapsids 22 .…”

“…S5 ) with 50 base pairs (bp) overlaps (Table S2) . The four DNA parts were mixed at an equimolar concentration in the nanomolar range and annealed according to a published protocol 23 . The assembly reaction was directly added to the TXTL reaction to obtain T7 WT 10 10 - 10 11 PFU/ml titers, similar to performing TXTL with a T7 WT genome concentration of 0.1 nM (Fig.…”

“…PHEIGES presents several advantages compared with ORACLE 20 and other current methods 22 23 16 62 in generating and identifying phages with improved host ranges. Firstly, the library approach to generate infectious phages can be performed on any large genetic parts of the phage within a day as compared to ORACLE that necessitates additional cloning and downstream selection steps.…”

“…On the other hand, in vitro genome engineering is emerging as a complementary approach to assemble and edit bacteriophages with novel properties. In vitro (Gibson 20 or Golden gate 21 ) assemblies of bacteriophage genomes from PCR fragments were expressed in bacteria 21 , packaged in pro-capsids 22 , or rebooted in cell-free transcription-translation (TXTL) systems 20 . Despite their simplicity, cellular transformation and amplification induce bias and complexify downstream selection processes while genome packaging in procapsids limits the variety of genomes to engineer.…”

High-throughput nanopore DNA sequencing of large insert fosmid clones directly from bacterial colonies

“…For the remaining two contigs, Sanger sequencing yielded a double sequence pattern, suggesting the fosmid backbone-specific primers had multiple priming sites. We hypothesize that these fosmids either contained two pCC1-FOS backbones (the result of a cos site bypass where cloned inserts are too short) or that two different fosmids were present in the same cell (Supplementary figure 4) (23). Reproducibility of the phiXXer assembly pipeline was also evaluated.…”