Combining Genes from Multiple Phages for Improved Cell Lysis and DNA Transfer from Escherichia coli to Bacillus subtilis.

Mario Juhas,James W Ajioka,Christine Wong,Adam Driks

doi:10.1371/journal.pone.0165778

Abstract

The ability to efficiently and reliably transfer genetic circuits between the key synthetic biology chassis, such as Escherichia coli and Bacillus subtilis, constitutes one of the major hurdles of the rational genome engineering. Using lambda Red recombineering we integrated the thermosensitive lambda repressor and the lysis genes of several bacteriophages into the E. coli chromosome. The lysis of the engineered autolytic cells is inducible by a simple temperature shift. We improved the lysis efficiency by introducing different combinations of lysis genes from bacteriophages lambda, ΦX174 and MS2 under the control of the thermosensitive lambda repressor into the E. coli chromosome. We tested the engineered autolytic cells by transferring plasmid and bacterial artificial chromosome (BAC)-borne genetic circuits from E. coli to B. subtilis. Our engineered system combines benefits of the two main synthetic biology chassis, E. coli and B. subtilis, and allows reliable and efficient transfer of DNA edited in E. coli into B. subtilis.

Highlights

The ability to efficiently and reliably transfer genetic circuits between different synthetic biology chassis, such as Escherichia coli and Bacillus subtilis, constitutes one of the main bottlenecks of the rational genome engineering
The lysis gene cassette of the phage lambda consists of four genes, namely S, R, Rz and Rz1 regulated by pR promoter
As the presence of an entire phage could be detrimental to the quality of the transferred DNA we aimed to efficiently lyse E. coli cells using solely the lysis genes from multiple bacteriophages

Summary

Introduction

The ability to efficiently and reliably transfer genetic circuits between different synthetic biology chassis, such as Escherichia coli and Bacillus subtilis, constitutes one of the main bottlenecks of the rational genome engineering. The Gram-negative E. coli and the Gram-positive B. subtilis are both well-characterized bacteria used in a number of synthetic biology and biotechnology applications [1,2,3,4]. They are considered to be promising chassis for the construction of the minimal cell factories [1, 5,6,7]. E. coli was successfully engineered for the production of a number of industrially relevant products, such as biofuels, amino acids and isoprenoids [2, 8,9,10], B. subtilis is considered to be a better host for certain applications [11,12,13]. B. subtilis secretes proteins into the medium and forms durable endospores

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Results

Conclusion