Hedenstierna K O, Lee Y H, Yang Y, Fox G E
Department of Biochemical and Biophysical Sciences, University of Houston, TX 77204-5934, USA.
Syst Appl Microbiol. 1993;16:280-6. doi: 10.1016/s0723-2020(11)80481-9.
A prototype stable RNA identification cassette for monitoring genetically engineered plasmids carried by strains of Escherichia coli has been developed. The cassette consists of a Vibrio proteolyticus 5S ribosomal RNA (rRNA) gene surrounded by promoters and terminators from the rrnB operon of Escherischia coli. The identifier RNA is expressed and successfully processed so that approximately 30% of the 5S rRNA isolated from either whole cells or 70S ribosomes is of the V. proteolyticus type. Cells carrying the identifier are readily detectable by hybridization. Accurate measurements show that the identification cassette has little effect on fitness compared to a strain containing an analogous plasmid carrying wild type E. coli 5S rRNA, and the V. proteolyticus 5S rRNA gene is not inactivated after prolonged growth. These results demonstrate the feasibility of developing small standardized identification cassettes that can utilize already existing highly sensitive rRNA detection methods. Cassettes of this type could in principle be incorporated into either the engineered regions of recombinant plasmids or their hosts.
一种用于监测大肠杆菌菌株携带的基因工程质粒的稳定RNA鉴定盒原型已被开发出来。该鉴定盒由一个溶蛋白弧菌5S核糖体RNA（rRNA）基因组成，其周围是来自大肠杆菌rrnB操纵子的启动子和终止子。鉴定RNA被表达并成功加工，使得从全细胞或70S核糖体中分离出的5S rRNA约30%是溶蛋白弧菌类型。携带该鉴定物的细胞可通过杂交轻易检测到。精确测量表明，与含有携带野生型大肠杆菌5S rRNA的类似质粒的菌株相比，该鉴定盒对适应性影响很小，并且溶蛋白弧菌5S rRNA基因在长时间生长后不会失活。这些结果证明了开发小型标准化鉴定盒的可行性，这些鉴定盒可以利用现有的高度灵敏的rRNA检测方法。这种类型的鉴定盒原则上可以整合到重组质粒的工程区域或其宿主中。
