Szafranski P, Mello C M, Sano T, Smith C L, Kaplan D L, Cantor C R
Department of Biomedical Engineering, Boston University, MA 02215, USA.
Proc Natl Acad Sci U S A. 1997 Feb 18;94(4):1059-63. doi: 10.1073/pnas.94.4.1059.
The use of microorganisms in the open environment would be of less concern if they were endowed with programmed self-destruction mechanisms. Here, we propose a new genetic design to increase the effectiveness of cell suicide systems. It ensures very tight control of the derepression of cell death by the combination of the bacteriophage T7 RNA polymerase-lysozyme system and an inducible synthesis of antisense RNA and the Escherichia coli LacI repressor. Functionality of this regulatory concept was tested by applying it to containment of Gram-negative bacteria, based on the conditional expression of the lethal Streptomyces avidinii streptavidin gene. Toxicity of streptavidin is derived from its exceptionally high binding affinity for an essential prosthetic group, D-biotin. The entire construct was designed to allow the soil bacterium Pseudomonas putida to survive only in the presence of aromatic hydrocarbons and their derivatives which it can degrade. Under favorable growth conditions, clones escaping killing appeared at frequencies of only 10(-7)-10(-8) per cell per generation. The general requirement for biotin through the living world should make streptavidin-based conditional lethal designs applicable to a broad range of containment strategies.
如果微生物具备程序性自我毁灭机制，那么在开放环境中使用它们就会较少受到关注。在此，我们提出一种新的基因设计，以提高细胞自杀系统的有效性。它通过噬菌体T7 RNA聚合酶-溶菌酶系统与反义RNA的诱导合成以及大肠杆菌LacI阻遏物的组合，确保对细胞死亡去阻遏进行非常严格的控制。通过将这种调控概念应用于革兰氏阴性菌的遏制，基于致死性阿维链霉菌抗生物素蛋白基因的条件性表达，对其功能进行了测试。抗生物素蛋白的毒性源于其对必需辅基D-生物素的极高结合亲和力。整个构建体的设计使得土壤细菌恶臭假单胞菌仅在存在其能够降解的芳烃及其衍生物时才能存活。在有利的生长条件下，逃避杀伤的克隆出现频率仅为每代每个细胞10^(-7)-10^(-8)。整个生物界对生物素的普遍需求应使基于抗生物素蛋白的条件致死设计适用于广泛的遏制策略。
