Rapid 40 kb genome construction from 52 parts

ABSTRACTLarge DNA constructs (>10 kb), including small genomes and artificial chromosomes, are invaluable tools for genetic engineering and vaccine development. However, the manufacture of these constructs is laborious. To address this problem, we applied new design insights and modified protocols to Golden Gate assembly. While this methodology is routinely used to assemble 5-10 DNA parts in one-step, we found that optimized assembly permitted >50 DNA fragments to be faithfully assembled in a single reaction. We applied these insights to genome construction, carrying out rapid assembly of the 40 kb T7 bacteriophage genome from 52 parts and recovering infectious phage particles after cellular transformation. The new Golden Gate assembly protocols and design principles described here can be applied to rapidly engineer a wide variety of large and complex assembly targets.

Motley Crew: Overview of the Currently Available Phage Diversity

The first complete genome that was sequenced at the beginning of the sequencing era was that of a phage, since then researchers throughout the world have been steadily describing and publishing genomes from a wide array of phages, uncovering the secrets of the most abundant and diverse biological entities known to man. Currently, we are experiencing an unprecedented rate of novel bacteriophage discovery, which can be seen from the fact that the amount of complete bacteriophage genome entries in public sequence repositories has more than doubled in the past 3 years and is steadily growing without showing any sign of slowing down. The amount of publicly available phage genome-related data can be overwhelming and has been summarized in literature before but quickly becomes out of date. Thus, the aim of this paper is to briefly outline currently available phage diversity data for public acknowledgment that could possibly encourage and stimulate future “depth” studies of particular groups of phages or their gene products.

A Method for Improving the Accuracy and Efficiency of Bacteriophage Genome Annotation

Bacteriophages are the most numerous entities on Earth. The number of sequenced phage genomes is approximately 8000 and increasing rapidly. Sequencing of a genome is followed by annotation, where genes, start codons, and functions are putatively identified. The mainstays of phage genome annotation are auto-annotation programs such as Glimmer and GeneMark. Due to the relatively small size of phage genomes, many groups choose to manually curate auto-annotation results to increase accuracy. An additional benefit of manual curation of auto-annotated phage genomes is that the process is amenable to be performed by students, and has been shown to improve student recruitment to the sciences. However, despite its greater accuracy and pedagogical value, manual curation suffers from high labor cost, lack of standardization and a degree of subjectivity in decision making, and susceptibility to mistakes. Here, we present a method developed in our lab that is designed to produce accurate annotations while reducing subjectivity and providing a degree of standardization in decision-making. We show that our method produces genome annotations more accurate than auto-annotation programs while retaining the pedagogical benefits of manual genome curation.