进化稳定微生物杀伤开关的合理设计
Rational Design of Evolutionarily Stable Microbial Kill Switches.
作者信息
Stirling Finn, Bitzan Lisa, O'Keefe Samuel, Redfield Elizabeth, Oliver John W K, Way Jeffrey, Silver Pamela A
机构信息
Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Warren Alpert 536, Boston, MA 02115, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, 3 Blackfan Circle, 5th Floor, Boston, MA 02115, USA.
Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Warren Alpert 536, Boston, MA 02115, USA.
出版信息
Mol Cell. 2017 Nov 16;68(4):686-697.e3. doi: 10.1016/j.molcel.2017.10.033.
The evolutionary stability of synthetic genetic circuits is key to both the understanding and application of genetic control elements. One useful but challenging situation is a switch between life and death depending on environment. Here are presented "essentializer" and "cryodeath" circuits, which act as kill switches in Escherichia coli. The essentializer element induces cell death upon the loss of a bi-stable cI/Cro memory switch. Cryodeath makes use of a cold-inducible promoter to express a toxin. We employ rational design and a toxin/antitoxin titering approach to produce and screen a small library of potential constructs, in order to select for constructs that are evolutionarily stable. Both kill switches were shown to maintain functionality in vitro for at least 140 generations. Additionally, cryodeath was shown to control the growth environment of a population, with an escape frequency of less than 1 in 10 after 10 days of growth in the mammalian gut.
合成基因回路的进化稳定性对于理解和应用基因控制元件至关重要。一种有用但具有挑战性的情况是根据环境在生死之间进行切换。本文介绍了“必需化器”和“冷冻死亡”回路，它们在大肠杆菌中充当致死开关。必需化器元件在双稳态cI/Cro记忆开关丧失时诱导细胞死亡。冷冻死亡利用冷诱导启动子来表达毒素。我们采用合理设计和毒素/抗毒素滴定方法来产生和筛选一小批潜在构建体文库，以便选择具有进化稳定性的构建体。两种致死开关在体外至少140代都保持了功能。此外，冷冻死亡被证明可以控制群体的生长环境，在哺乳动物肠道中生长10天后逃逸频率低于十分之一。
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