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Liposome fragment-mediated introduction of multiple plasmids into Bacillus subtilis.

ABSTRACT: Transformation of microorganisms by plasmid introduction is one of the central techniques in modern biotechnology. However, applicable transformation methods for simultaneous introduction of multiple plasmids are still limiting. Here, we reported a liposome-mediated method that efficiently introduces multiple plasmids into B. subtilis. In this method, liposomes containing three kinds of plasmids were mixed with B. subtilis protoplasts in the presence of 36% polyethylene glycol (PEG), and the resultant protoplasts were grown in cell wall-regeneration media. We found that the rates of introduction of multiple plasmids were significantly increased in the presence of liposomes. We also found that an intact liposome structure was not required for introduction, and the presence of phosphatidylglycerol (PG) was important for efficient introduction of multiple plasmids. Therefore, the liposome- or liposome fragment-mediated transformation method reported here can advance studies utilizing multiple plasmids.

SUBMITTER: Ohta K

PROVIDER: S-EPMC6512750 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Liposome fragment-mediated introduction of multiple plasmids into Bacillus subtilis.

Ohta Kazuki K Ichihashi Norikazu N

Biochemistry and biophysics reports 20190508

Transformation of microorganisms by plasmid introduction is one of the central techniques in modern biotechnology. However, applicable transformation methods for simultaneous introduction of multiple plasmids are still limiting. Here, we reported a liposome-mediated method that efficiently introduces multiple plasmids into B. subtilis. In this method, liposomes containing three kinds of plasmids were mixed with B. subtilis protoplasts in the presence of 36% polyethylene glycol (PEG ...[more]

PMID: 31111103

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Project description:RNA-DNA hybrids are common in chromosomal DNA. Persistent RNA-DNA hybrids result in replication fork stress, DNA breaks, and neurological disorders in humans. During replication, Okazaki fragment synthesis relies on frequent RNA primer placement, providing one of the most prominent forms of covalent RNA-DNA strands in vivo The mechanism of Okazaki fragment maturation, which involves RNA removal and subsequent DNA replacement, in bacteria lacking RNase HI remains unclear. In this work, we reconstituted repair of a linear model Okazaki fragment in vitro using purified recombinant enzymes from Bacillus subtilis We showed that RNase HII and HIII are capable of incision on Okazaki fragments in vitro and that both enzymes show mild stimulation by single-stranded DNA binding protein (SSB). We also showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturation in vitro Furthermore, we found that YpcP is a 5' to 3' nuclease that can act on a wide variety of RNA- and DNA-containing substrates and exhibits preference for degrading RNA in model Okazaki fragments. Together, our data showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturation, whereas YpcP also contributes to the removal of RNA from an Okazaki fragment in vitroIMPORTANCE All cells are required to resolve the different types of RNA-DNA hybrids that form in vivo When RNA-DNA hybrids persist, cells experience an increase in mutation rate and problems with DNA replication. Okazaki fragment synthesis on the lagging strand requires an RNA primer to begin synthesis of each fragment. The mechanism of RNA removal from Okazaki fragments remains unknown in bacteria that lack RNase HI. We examined Okazaki fragment processing in vitro and found that RNase HIII in conjunction with DNA polymerase I represent the most efficient repair pathway. We also assessed the contribution of YpcP and found that YpcP is a 5' to 3' exonuclease that prefers RNA substrates with activity on Okazaki and flap substrates in vitro.

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Dataset Information

Liposome fragment-mediated introduction of multiple plasmids into Bacillus subtilis.

Publications

Liposome fragment-mediated introduction of multiple plasmids into <i>Bacillus subtilis</i>.

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