Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana-Cuajimalpa, Vasco de Quiroga 4871, Santa Fe, 05348, Mexico City, Mexico.
Posgrado en Ciencias Naturales e Ingeniería, Universidad Autónoma Metropolitana-Cuajimalpa, Vasco de Quiroga 4871, Santa Fe, 05348, Mexico City, Mexico.
Bioprocess Biosyst Eng. 2019 Aug;42(8):1391-1397. doi: 10.1007/s00449-019-02129-2. Epub 2019 Apr 20.
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) 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 values, pminiR1 was introduced in an engineered E. coli strain expressing the Vitreoscilla hemoglobin inserted in chromosome (W12). The Y 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 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.
开发了一种由 R1 复制子的最小复制控制元件和卡那霉素抗性标记组成的合成质粒，命名为 pminiR1。在有氧和微氧条件下，在大肠杆菌 W3110 recA（W1）中测试了 pminiR1 的产生。在有氧和微氧条件下，质粒 DNA 产量分别仅为生物量（Y）的 0.06±0.02 和 0.22±0.11mg/g。作为增加 Y 值的一种选择，将 pminiR1 引入在染色体中表达插入的玻璃滑液血红蛋白的工程大肠杆菌菌株（W12）中。使用菌株 W12 的 Y 值分别增加到有氧和微氧条件下的 0.85±0.05 和 1.53±0.14mg/g。在有氧和微氧条件下，在 37°C 下比较了两种菌株中 pminiR1 和 pUC57Kan 的产生。使用菌株 W12 的 pminiR1 的 Y 值分别为有氧和微氧条件下的 6.25±0.16 和 9.27±0.95mg/g。这些产量与使用菌株 W12 的质粒 pUC57Kan 获得的产量相似（有氧和微氧培养物的 6.9±0.64 和 10.85±1.06mg/g）。因此，基于 R1 复制子的合成最小质粒是生物技术应用中 pUC 质粒的有价值替代品。
