PDF(Pubmed)
Abstract：
A major challenge in the fields of biological imaging and synthetic biology is noninvasively visualizing the functions of natural and engineered cells inside opaque samples such as living animals. One promising technology that addresses this limitation is ultrasound (US), with its penetration depth of several cm and spatial resolution on the order of 100 μm. Within the past decade, reporter genes for US have been introduced and engineered to link cellular functions to US signals via heterologous expression in commensal bacteria and mammalian cells. These acoustic reporter genes (ARGs) represent a novel class of genetically encoded US contrast agent, and are based on air-filled protein nanostructures called gas vesicles (GVs). Just as the discovery of fluorescent proteins was followed by the improvement and diversification of their optical properties through directed evolution, here we describe the evolution of GVs as acoustic reporters. To accomplish this task, we establish high-throughput, semiautomated acoustic screening of ARGs in bacterial cultures and use it to screen mutant libraries for variants with increased nonlinear US scattering. Starting with scanning site saturation libraries for two homologues of the primary GV structural protein, GvpA/B, two rounds of evolution resulted in GV variants with 5- and 14-fold stronger acoustic signals than the parent proteins. We anticipate that this and similar approaches will help high-throughput protein engineering play as large a role in the development of acoustic biomolecules as it has for their fluorescent counterparts.
摘要：
生物成像和合成生物学领域的主要挑战是非侵入性可视化不透明样品(例如活体动物)内的天然和工程化细胞的功能。解决这一限制的一种有前途的技术是超声波（US），其穿透深度为几厘米，空间分辨率约为100μm。在过去的十年里，US的报道基因已经被引入和工程改造以通过共生细菌和哺乳动物细胞中的异源表达将细胞功能与US信号联系起来。这些声学报告基因（ARGs）代表了一类新的基因编码的US造影剂，并且基于被称为气体囊泡（GVs）的充满空气的蛋白质纳米结构。正如荧光蛋白的发现之后，通过定向进化其光学特性的改进和多样化，在这里，我们将GV的演变描述为声学记者。为了完成这项任务,我们建立了高通量，在细菌培养物中对ARG进行半自动声学筛选，并使用它来筛选突变文库中非线性US散射增加的变体。从扫描主要GV结构蛋白的两个同源物的位点饱和文库开始，GvpA/B,两轮进化导致GV变体具有比亲本蛋白强5倍和14倍的声学信号。我们预计这种方法和类似的方法将有助于高通量蛋白质工程在声学生物分子的开发中发挥与其荧光对应物一样大的作用。
