Kobayashi Hideki
Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Japan
Appl Environ Microbiol. 2015 Apr;81(7):2544-53. doi: 10.1128/AEM.03708-14. Epub 2015 Jan 30.
Recently, artificial gene networks have been developed in synthetic biology to control gene expression and make organisms as controllable as robots. Here, I present an artificial posttranslational gene-silencing system based on the codon usage bias and low tRNA content corresponding to minor codons. I engineered the green fluorescent protein (GFP) gene to inhibit translation indirectly with the lowest-usage codons to monopolize various minor tRNAs (lgfp). The expression of lgfp interfered nonspecifically with the growth of Escherichia coli, Saccharomyces cerevisiae, human HeLa cervical cancer cells, MCF7 breast cancer cells, and HEK293 kidney cells, as well as phage and adenovirus expansion. Furthermore, insertion of lgfp downstream of a phage response promoter conferred phage resistance on E. coli. Such engineered gene silencers could act as components of biological networks capable of functioning with suitable promoters in E. coli, S. cerevisiae, and human cells to control gene expression. The results presented here show general suppressor artificial genes for live cells and viruses. This robust system provides a gene expression or cell growth control device for artificially synthesized gene networks.
最近,合成生物学领域开发了人工基因网络来控制基因表达,使生物体像机器人一样可控。在此,我展示了一种基于密码子使用偏好和与稀有密码子相对应的低tRNA含量的人工翻译后基因沉默系统。我对绿色荧光蛋白(GFP)基因进行了改造,使其用最低使用频率的密码子间接抑制翻译,从而独占各种稀有tRNA(lgfp)。lgfp的表达非特异性地干扰了大肠杆菌、酿酒酵母、人宫颈癌细胞HeLa、人乳腺癌细胞MCF7和人肾细胞HEK293的生长,以及噬菌体和腺病毒的扩增。此外,在噬菌体反应启动子下游插入lgfp赋予了大肠杆菌对噬菌体的抗性。这种经过工程改造的基因沉默子可以作为生物网络的组件,能够与合适的启动子一起在大肠杆菌、酿酒酵母和人类细胞中发挥作用以控制基因表达。此处展示的结果表明存在针对活细胞和病毒的通用抑制性人工基因。这个强大的系统为人工合成基因网络提供了一种基因表达或细胞生长控制装置。