T7 RNA polymerase-driven inducible cell lysis for DNA transfer from Escherichia coli to Bacillus subtilis.

Mario Juhas,James W Ajioka

doi:10.1111/1751-7915.12843

Abstract

SummaryThe majority of the good DNA editing techniques have been developed in Escherichia coli; however, Bacillus subtilis is better host for a plethora of synthetic biology and biotechnology applications. Reliable and efficient systems for the transfer of synthetic DNA between E. coli and B. subtilis are therefore of the highest importance. Using synthetic biology approaches, such as streamlined lambda Red recombineering and Gibson Isothermal Assembly, we integrated genetic circuits pT7L123, Repr‐ts‐1 and pLT7pol encoding the lysis genes of bacteriophages MS2, ΦX174 and lambda, the thermosensitive repressor and the T7 RNA polymerase into the E. coli chromosome. In this system, T7 RNA polymerase regulated by the thermosensitive repressor drives the expression of the phage lysis genes. We showed that T7 RNA polymerase significantly increases efficiency of cell lysis and transfer of the plasmid and bacterial artificial chromosome‐encoded DNA from the lysed E. coli into B. subtilis. The T7 RNA polymerase‐driven inducible cell lysis system is suitable for the efficient cell lysis and transfer of the DNA engineered in E. coli to other naturally competent hosts, such as B. subtilis.

Highlights

The Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Bacillus subtilis are among the key cellular chassis suitable for a plethora of synthetic biology devices and biotechnology applications (Harwood and Cranenburgh, 2008; Ajikumar et al, 2010; Commichau et al, 2014; Juhas and Ajioka, 2016)
We showed previously that combining lysis genes of the bacteriophages MS2, ΦX174 and lambda in the same cell significantly improves lysis efficiency of E. coli cells (Juhas et al, 2016)
Lysis genes of the bacteriophages MS2, ΦX174 and lambda in the engineered genetic circuit pT7L123 are located downstream of the pT7 promoter, which is regulated by the T7 RNA polymerase

Summary

The majority of the good DNA editing techniques have been developed in Escherichia coli; Bacillus subtilis is better host for a plethora of synthetic biology and biotechnology applications. Using synthetic biology approaches, such as streamlined lambda Red recombineering and Gibson Isothermal Assembly, we integrated genetic circuits pT7L123, Repr-ts-1 and pLT7pol encoding the lysis genes of bacteriophages MS2, ΦX174 and lambda, the thermosensitive repressor and the T7 RNA polymerase into the E. coli chromosome. In this system, T7 RNA polymerase regulated by the thermosensitive repressor drives the expression of the phage lysis genes. The T7 RNA polymerase-driven inducible cell lysis system is suitable for the efficient cell lysis and transfer of the DNA engineered in E. coli to other naturally competent hosts, such as B. subtilis

Introduction

Results and discussion

Conclusions

Experimental procedures