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首页> 外文期刊>Journal of Biotechnology >Deletion formation mutations in plasmid expression vectors are unfavored by runaway amplification conditions and differentially selected under kanamycin stress
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Deletion formation mutations in plasmid expression vectors are unfavored by runaway amplification conditions and differentially selected under kanamycin stress

机译：失控扩增条件不利于质粒表达载体中的缺失形成突变，并在卡那霉素胁迫下进行差异选择

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Plasmid pCIneo is a ColE1-like mammalian expression vector also used as backbone for DNA vaccine development. We have recently shown that pCIneo spontaneously recombines due to the presence of two 28bp direct repeats. The persistence of low-frequency recombinants led us to evaluate the impact of environmental stresses typically found during plasmid production on plasmid copy number and recombination frequency. We observed an increase in pCIneo amplification (2.6-4.3-fold) in Escherichia coli cultures grown at 42 degrees C and also in minimal medium (at both 37 degrees C and 42 degrees C). These conditions fit to the smallest ratio between recombinant molecules and total plasmids. Conversely, increasing the dissolved oxygen tension from 20% to 40% in rich media did not have a significant impact on both plasmid copy number and recombination frequency, independently of the temperature used. We have also shown recently that the neomycin resistance (neo(r)) gene of pCIneo becomes actively transcribed as a result of recombination between the repeats. This prompted us to gain some insight into plasmid adaptation and competition by evaluating the impact of distinct concentrations of kanamycin on the differential selection of plasmid recombinant forms: monomer and heterodimers (1+2 and 1+3). We found the monomeric form to be predominantly recovered at lower concentrations of antibiotic whilst higher concentrations led to an increase in the percentage of the 1+2 form. The 1+3 heterodimeric form was invariably found at low percentages, independently of the concentration used.
机译：质粒pCIneo是一种类似于ColE1的哺乳动物表达载体，也被用作DNA疫苗开发的骨架。我们最近发现，由于存在两个28bp的直接重复序列，pCIneo自发重组。低频重组体的持续存在使我们评估了通常在质粒生产过程中发现的环境胁迫对质粒拷贝数和重组频率的影响。我们观察到在42摄氏度以及在基本培养基（37摄氏度和42摄氏度）下生长的大肠杆菌培养物中pCIneo扩增的增加（2.6-4.3倍）。这些条件适合重组分子与总质粒之间的最小比例。相反，将富氧培养基中的溶解氧张力从20％增加到40％对质粒拷贝数和重组频率均无显着影响，与所用温度无关。最近我们还显示，pCIneo的新霉素抗性（neo（r））基因由于重复序列之间的重组而变得活跃。这促使我们通过评估不同浓度的卡那霉素对质粒重组形式（单体和异二聚体（1 + 2和1 + 3））的差异选择的影响来获得对质粒适应和竞争的一些见解。我们发现单体形式主要在较低的抗生素浓度下被回收，而较高的浓度则导致1 + 2形式的百分比增加。总是以低百分比找到1 + 3异二聚体形式，与所用浓度无关。

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