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

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.

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

“…We chose lysis genes of the bacteriophages MS2, ΦX174 and lambda mainly because they have different mechanism of action and are all well characterized (Young, ; Gründling et al ., ; Berry et al ., ; Shulman et al ., ; Tanaka and Clemons, ; Catalão et al ., ). Furthermore, 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 ., ). Lysis genes of the bacteriophages MS2, ΦX174 and lambda in the engineered genetic circuit pT7 L123 are located downstream of the pT7 promoter, which is regulated by the T7 RNA polymerase.…”

“…pJScav, iBAC(cav)1 and iBAC(10) transferred into the recipient B. subtilis with various efficiency for the donor E. coli strains Ec(Ri), Ec(R pT7 L123 pL T7i) and Ec(R pL L123i). When using strain Ec(Ri), the low‐efficiency transfer of pJScav and iBAC(cav)1 and iBAC(10) from E. coli to B. subtilis occurred due to spontaneous lysis of E. coli Ec(Ri) cells (Corchero et al ., ; Juhas et al ., ). Both donor strains Ec(R pT7 L123 pL T7i) and Ec(R pL L123i) transferred pJScav and iBAC(cav)1 and iBAC(10) to B. subtilis with higher efficiency than the strain Ec(Ri).…”

“…The strain Ec(R pL L123i) has the genetic circuits Repr‐ts‐1 and pL L123 integrated into the chromosome, but does not harbour the genetic circuit pL T7pol. Consequently, as the strain Ec(R pL L123i) has not been augmented with the genetic circuit pL T7pol, the expression of the lysis genes of the bacteriophages MS2, ΦX174 and lambda in the strain Ec(R pL L123i) is directly regulated by the thermosensitive repressor (Juhas et al ., ) instead of through T7 RNA polymerase.…”

“…As both E. coli and B. subtilis are among the main chassis for a number of synthetic biology and biotechnology applications, it is important to develop reliable and efficient systems for the transfer of synthetic DNA (especially the high‐molecular‐weight DNA, which is prone to physical breakage) between these two cell types. The previously engineered cell lysis systems either required the chemical inducer, enzyme or mechanical force or utilized the entire prophages to disrupt the donor E. coli cell, or their efficiency of lysis was not very high (Itaya and Kaneko, ; Kaneko and Itaya, ; Gao et al ., ; Juhas et al ., ).…”

“…In this system, the co‐culture method was used to transfer the high‐molecular‐weight DNA from E. coli undergoing lambda prophage‐induced lysis to B. subtilis (Itaya and Kaneko, ; Kaneko and Itaya, ). Furthermore, transfer of genetic circuits from the lysed E. coli to B. subtilis , which does not rely on the entire phage to lyse the donor cell, has been demonstrated recently (Juhas et al ., ). This system utilizes only the individual lysis genes from multiple phages to disrupt the donor E. coli cell.…”