Effect of plasmid copy number and lac operator sequence on antibiotic-free plasmid selection by operator-repressor titration in Escherichia coli

Journal of Molecular Microbiology and Biotechnology
Rocky M CranenburghJulian A J Hanak

Abstract

The Escherichia coli strain DH1lacdapD enables plasmid selection and maintenance that is free from antibiotics and selectable marker genes. This is achieved by using only the lac operator sequence as a selectable element. This strain is currently used to generate high copy number plasmids with no antibiotic resistance genes for use as DNA vaccines and for expression of recombinant proteins. Until now these have been limited to pUC-based plasmids containing a high copy number pMB1-derived origin of replication, and the principle lacO(1) and auxiliary lacO(3) operators. In this study we have shown that this system can also be used to select and maintain pBR322-based plasmids with the lower copy number pMB1 origin of replication, and that lacO(1) alone or a palindromic version of lacO(1) can provide a sufficient level of repressor titration for plasmid selection. This is advantageous for recombinant protein production, where low copy number plasmids are often used and plasmid maintenance is important. The degree of repressor titration due to these plasmids was measured using the natural lactose operon in E. coli DH1 as a model.

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Citations

Feb 12, 2009·Applied Microbiology and Biotechnology·Diana M Bower, Kristala L J Prather
Oct 5, 2007·Molecular Biotechnology·Elisabete CarapuçaFilipe J M Mergulhão
May 7, 2009·Immunology and Cell Biology·James E GalenMyron M Levine
Apr 10, 2015·Pathogens·Charlotte MignonBettina Werle
Mar 23, 2013·Microbiology·Oliver WrightTom Ellis
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Jan 10, 2018·Bioprocess and Biosystems Engineering·Jiali XuHe Huang
Sep 7, 2007·Biotechnology Journal·Jürgen MairhoferReingard Grabherr
Jan 25, 2011·Microbial Biotechnology·Jens KrollAlexander Steinbüchel
Jul 19, 2019·Clinical and Experimental Immunology·R R C New
Feb 15, 2013·Human Vaccines & Immunotherapeutics·Jin Yuan WangJames E Galen
May 7, 2021·Essays in Biochemistry·Aatir A TungekarLloyd W Ruddock

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