Abstract:

With the rapid rise of synthetic genes and engineered bacteria in disease diagnosis and treatment, it poses a growing risk of biosafety issues. We review the biocontainment strategies based on synthetic biology, and especially highlight the recent biocontainment studies on diagnosis or therapeutic bacteria. There are several goals of biocontainment. One is to reduce the escapee rate of engineered bacteria by limiting the survival within biological barriers. The second is to prevent synthetic genes transferring from engineered bacteria into environment. Auxotrophy and kill-switches are widely applied in biocontainment of engineered bacteria. Auxotrophic organisms with essential genes knockout rely on key metabolites supplement for survival. Kill-switches are inducible toxic gene circuits, such as suicide switch and toxin-antitoxin system. Once engineered bacteria leave from human bodies, the toxic genes are switched on to kill organisms. Genetic separation and DNA breaking are useful strategies to keep synthetic genes from spreading into environmental organisms. Essential genes and genes of interest can be distributed into multiple vectors or chromosomes, each vector or chromosome depends on the others for replication. DNA breaking technologies like CRISPR or other DNA nucleases are used to digest chromosome or plasmid inside engineered bacteria, which regulates host survival and synthetic gene transferring. The cross-feeding of environmental metabolites and genes into engineered bacteria could lead to biocontainment failure. Several unnatural nucleic acids have been developed for gene replication and transcription, and much more unnatural amino acids are deployed for protein translation. One advantage of these unnatural systems is the orthogonality, which prevent synthetic gene transfer to natural organism. The chemically synthesized unnatural nucleic acid and amino acids are not present in environment, so the synthetic auxotrophy overcomes the cross-feeding limitation of natural auxotrophy. Biocontainment systems with multiple-layered design based on different synthetic biological principles have the potentials to solve the biosafety issues in the future.

Key words: biocontainment, auxotrophy, suicide gene, toxin-antitoxin, gene editing, unnatural nucleic acid, unnatural amino acid

摘要：

随着人工设计的基因元件和工程菌应用于医学诊断和疾病治疗领域的增加，由此产生的生物安全风险也越来越受到重视。本文主要回顾了合成生物学的生物安全防控策略，特别介绍了近几年医学诊疗工程菌的生物安全防控研究。工程菌的生物安全防控可以防止宿主菌和基因元件脱离病灶区域向环境泄漏。基于营养缺陷或自杀基因的调控系统广泛用于限制工程菌的逃逸，基因元件拆分和靶向降解策略则可以防止基因元件扩散到环境中被其他细胞利用。环境中的代谢物和基因片段可能转移进入工程菌，这是导致生物安全防控机制失效的重要因素。非天然核苷酸和非天然氨基酸等非天然复制翻译系统的正交性好，可以大幅减少环境和工程菌间的相互影响。综合不同合成生物学原理设计的多层生物安全防控系统对于未来解决生物安全问题具有极大潜力。

关键词: 生物安全防控, 营养缺陷, 自杀基因, 毒素-抗毒素, 基因编辑, 非天然核苷酸, 非天然氨基酸