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Design of a synthetic miniR1 plasmid and its production by engineered Escherichia coli

A synthetic plasmid consisting of the minimal elements for replication control of the R1 replicon and kanamycin resistance marker, which was named pminiR1, was developed. pminiR1 production was tested at 30 °C under aerobic and microaerobic conditions in Escherichia coli W3110 recA − (W1). The plasm...

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Published in:Bioprocess and biosystems engineering 2019-08, Vol.42 (8), p.1391-1397
Main Authors: Lara, Alvaro R., Velázquez, Daniela, Penella, Inés, Islas, Fabiola, González-De la Rosa, Claudia H., Sigala, Juan-Carlos
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Sigala, Juan-Carlos
description A synthetic plasmid consisting of the minimal elements for replication control of the R1 replicon and kanamycin resistance marker, which was named pminiR1, was developed. pminiR1 production was tested at 30 °C under aerobic and microaerobic conditions in Escherichia coli W3110 recA − (W1). The plasmid DNA yields from biomass ( Y pDNA/X ) were only 0.06 ± 0.02 and 0.22 ± 0.11 mg/g under aerobic and microaerobic conditions, respectively. As an option to increase Y pDNA/X values, pminiR1 was introduced in an engineered E. coli strain expressing the Vitreoscilla hemoglobin inserted in chromosome (W12). The Y pDNA/X values using strain W12 increased to 0.85 ± 0.05 and 1.53 ± 0.14 mg/g under aerobic and microaerobic conditions, respectively. pminiR1 production in both strains was compared with that of pUC57Kan at 37 °C under aerobic and microaerobic conditions. The Y pDNA/X values for pminiR1 using strain W12 were 6.25 ± 0.16 and 9.27 ± 0.95 mg/g under aerobic and microaerobic conditions, respectively. Such yields were similar to those obtained for plasmid pUC57Kan using strain W12 (6.9 ± 0.64 and 10.85 ± 1.06 mg/g for aerobic and microaerobic cultures, respectively). Therefore, the synthetic minimal plasmid based on the R1 replicon is a valuable alternative to pUC plasmids for biotechnological applications.
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The plasmid DNA yields from biomass ( Y pDNA/X ) were only 0.06 ± 0.02 and 0.22 ± 0.11 mg/g under aerobic and microaerobic conditions, respectively. As an option to increase Y pDNA/X values, pminiR1 was introduced in an engineered E. coli strain expressing the Vitreoscilla hemoglobin inserted in chromosome (W12). The Y pDNA/X values using strain W12 increased to 0.85 ± 0.05 and 1.53 ± 0.14 mg/g under aerobic and microaerobic conditions, respectively. pminiR1 production in both strains was compared with that of pUC57Kan at 37 °C under aerobic and microaerobic conditions. The Y pDNA/X values for pminiR1 using strain W12 were 6.25 ± 0.16 and 9.27 ± 0.95 mg/g under aerobic and microaerobic conditions, respectively. Such yields were similar to those obtained for plasmid pUC57Kan using strain W12 (6.9 ± 0.64 and 10.85 ± 1.06 mg/g for aerobic and microaerobic cultures, respectively). 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subjects Bacteria
Bacterial Proteins - biosynthesis
Bacterial Proteins - genetics
Biotechnology
Chemistry
Chemistry and Materials Science
Chromosomes, Bacterial - genetics
Chromosomes, Bacterial - metabolism
Deoxyribonucleic acid
DNA
E coli
Environmental Engineering/Biotechnology
Escherichia coli
Escherichia coli - genetics
Escherichia coli - metabolism
Food Science
Hemoglobin
Industrial and Production Engineering
Industrial Chemistry/Chemical Engineering
Kanamycin
Microorganisms, Genetically-Modified - genetics
Microorganisms, Genetically-Modified - metabolism
Plasmids
Plasmids - biosynthesis
Plasmids - genetics
Rapid Communication
RecA protein
Recombinant Proteins - biosynthesis
Recombinant Proteins - genetics
Truncated Hemoglobins - biosynthesis
Truncated Hemoglobins - genetics
title Design of a synthetic miniR1 plasmid and its production by engineered Escherichia coli
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