Vockley J G, Pène J J
School of Life and Health Sciences, University of Delaware, Newark 19716.
Biotechniques. 1990 Dec;9(6):680, 682-3.
Plasmid pUC18rspL is a 3.788-kilobase pair vector, derived from pUC18, pKK232-8 and pHSG664, which identifies promoter-bearing DNA fragments functional in StrA E. coli by activation of a promoterless streptomycin-sensitive gene cartridge (rspL). Expression of the plasmid-borne rspL gene leads to sensitivity dominance and death of the cell. Promoter-bearing DNA fragments can be cloned within a synthetic polylinker containing 12 unique restriction nuclease target sequences. After transformation of StrA E. coli TB1, ampicillin-resistant transformants are replica plated on medium containing ampicillin and streptomycin to identify promoters cloned in their functional orientation. These elements can be sequenced without additional subcloning steps from the M13 universal forward primer hybridization site located 5' of the polylinker in the pUC18 contribution. Transcriptional terminators are cloned 3' of the rspL gene to maintain a balance between transcription and replication when high signal strength promoters such as the E. coli Tac promoter are analyzed. pUC18rspL is used to clone transcriptional promoters from the extreme thermophile T. aquaticus. Promoter signal strength can be estimated by determining the extent of sensitivity dominance conferred to the host.
质粒pUC18rspL是一个3.788千碱基对的载体，由pUC18、pKK232 - 8和pHSG664衍生而来，它通过一个无启动子的链霉素敏感基因盒（rspL）的激活来鉴定在StrA大肠杆菌中具有功能的含启动子的DNA片段。质粒携带的rspL基因的表达导致细胞的敏感性显性和死亡。含启动子的DNA片段可以克隆到一个含有12个独特限制性核酸酶靶序列的合成多克隆位点内。将StrA大肠杆菌TB1转化后，对氨苄青霉素抗性转化子进行影印接种到含有氨苄青霉素和链霉素的培养基上，以鉴定以其功能方向克隆的启动子。这些元件可以从位于pUC18贡献部分多克隆位点5'端的M13通用正向引物杂交位点进行测序，而无需额外的亚克隆步骤。转录终止子克隆在rspL基因的3'端下游，以便在分析如大肠杆菌Tac启动子等高信号强度启动子时，维持转录和复制之间的平衡。pUC18rspL用于从嗜热栖热菌中克隆转录启动子。启动子信号强度可以通过确定赋予宿主的敏感性显性程度来估计。
