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Copy number control of the streptococcal plasmid pIP501 occurs at three levels.

ABSTRACT: Transcriptional analysis of the replication region of plasmid pIP501 has revealed three active promoters. The repR gene which is essential for pIP501 replication was transcribed from promoter pII. A small antisense RNA (136 nt, RNAIII) generated from promoter pIII was complementary to the leader region of the repR mRNA. Introduction of either point mutations or deletions into promoter pIII or RNAIII resulted in a 5-20fold increased plasmid copy number suggesting a negative regulatory function for RNAIII. The copR gene, the complete DNA and amino acid sequence of which is reported, was dispensable for pIP501 replication. However, deletion of the copR promoter pI and/or the copR coding sequence led to a 10-20fold increase in plasmid copy number. This effect was also observed when a -1 frameshift mutation was introduced into the CopR coding region. Mutations in copR and pIII/RNAIII were not additive. It is, therefore, proposed that both components act at the same level of copy number control most likely in a sequential way. A second level of copy number control was found to involve an inverted repeat structure upstream of and overlapping with promoter pII. Destruction of this repeat sequence by deletion caused an increase in copy number 2-3fold higher than that observed for either RNAIII or copR mutations. A working model is proposed how different components of pIP501 interact to regulate its copy number.

SUBMITTER: Brantl S

PROVIDER: S-EPMC310398 | biostudies-other | 1992 Feb

REPOSITORIES: biostudies-other

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Copy number control of the streptococcal plasmid pIP501 occurs at three levels.

Brantl S S Behnke D D

Nucleic acids research 19920201 3

Transcriptional analysis of the replication region of plasmid pIP501 has revealed three active promoters. The repR gene which is essential for pIP501 replication was transcribed from promoter pII. A small antisense RNA (136 nt, RNAIII) generated from promoter pIII was complementary to the leader region of the repR mRNA. Introduction of either point mutations or deletions into promoter pIII or RNAIII resulted in a 5-20fold increased plasmid copy number suggesting a negative regulatory function fo ...[more]

PMID: 1741273

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Project description:Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5? cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a straightforward and reliable method to quantify the plasmid copy number. Therefore we believe that the ddPCR designed in this study will be widely used for any plasmid copy number calculation in the future.

Dataset Information

Copy number control of the streptococcal plasmid pIP501 occurs at three levels.

Publications

Copy number control of the streptococcal plasmid pIP501 occurs at three levels.

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