Avdyusheva E F, Lopasteyska Ya A, Sharov T N, Teteryatnikova N N, Molchanova E V
Department of Molecular Biology and Genetics, Volgograd State Medical University, Ministry of Health of the Russian Federation, Volgograd, Russia.
Volgograd Research Anti-Plague Institute, Federal Service for Supervision of Consumer Right Protection and Human Welfare, Volgograd, Russia.
Bull Exp Biol Med. 2017 Aug;163(4):519-522. doi: 10.1007/s10517-017-3842-7. Epub 2017 Aug 29.
We demonstrated the possibility of obtaining insertion mutants by a modified technique using EZ::TN5 system during culturing of the recipient strain on a dense nutrient medium and exclusion of the centrifugation stage. The frequency of transposon mutants of E. coli 10979/EZ::TN5 was 2×10. Genetically modified strains were characterized by kanamycin resistance, inability to L-malate assimilation, changes in the expression of individual proteins of protein mass-spectra (5096.3, 6252.9, and 9067.7 Da), and the presence of fragments in genomic DNA amplified by specific forward and reverse primers that were homologous to Tn5 transposon insertion sites. The modified procedure for obtaining insertion mutants by using EZ::TN5 system was not inferior by the efficiency to the standard procedure, but shortens experiment duration, simplifies it, and reduces the risks related to working with group 2 pathogenicity microorganisms.
我们证明了在受体菌株于致密营养培养基上培养期间，通过使用EZ::TN5系统的改良技术并排除离心步骤来获得插入突变体的可能性。大肠杆菌10979/EZ::TN5的转座子突变体频率为2×10。基因修饰菌株的特征在于对卡那霉素具有抗性，无法同化L-苹果酸，蛋白质质谱中个别蛋白质（5096.3、6252.9和9067.7 Da）的表达发生变化，以及通过与Tn5转座子插入位点同源的特异性正向和反向引物扩增的基因组DNA中存在片段。使用EZ::TN5系统获得插入突变体的改良程序在效率上并不逊于标准程序，但缩短了实验持续时间，简化了实验，并降低了与处理2类致病性微生物相关的风险。
