Abstract
Plasmids are extra chromosomal DNA that can confer to their hosts' supplementary characteristics such as antibiotic resistance. Plasmids code for their copy number through their own replication frequency. Even though the biochemical networks underlying the plasmid copy number (PCN) regulation processes have been studied and modeled, no measurement of the heterogeneity in PCN within a whole population has been done. We have developed a fluorescent-based measurement system, which enables determination of the mean and noise in PCN within a monoclonal population of bacteria. Two different fluorescent protein reporters were inserted: one on the chromosome and the other on the plasmid. The fluorescence of these bacteria was measured with a microfluidic flow cytometry device. We show that our measurements are consistent with known plasmid characteristics. We find that the partitioning system lowers the PCN mean and standard deviation. Finally, bacterial populations were allowed to grow without selective pressure. In this case, we were able to determine the plasmid loss rate and growth inhibition effect.
Highlights
Plasmids are extra chromosomal DNA fragments that constitute as much as 25% of some organisms’ genetic material1͔
We modified the Top10 ͑Clontechstrain, which is a derivative of Escherichia coli K-12 as follows: chromosomal copy of lacI was replaced by lacIq1 allele18͔ by homologous recombination using the counterselection bacterial artificial chromosomeBACmodification kit from Genebridges19͔ ͑Genebridges GmbH, Dresden, Germany
In order to verify the linearity of expression with gene copy number, two mOrange expression systems were cloned in the mini-F plasmid
Summary
Plasmids are extra chromosomal DNA fragments that constitute as much as 25% of some organisms’ genetic material1͔. The genes coded on plasmids often transfer between different species, making them an interesting pool of shared genetic material2͔. In order to replicate themselves, plasmids use the replication machinery of their hosts. These features allow the plasmids to exhibit interesting symbiotic behaviors with their host. Plasmids code for a biochemical network that regulates their replication frequency4͔. This regulation network, which varies from plasmid to plasmid, sets the plasmid copy numberPCN. Researches on plasmids have focused on unraveling and understanding the networks controlling the replication frequency4͔. The use of noncoding RNA in regulation processes was first discovered in plasmids5͔
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