若干裂解基因盒子的表征及应用

Characterization and application of several lysis cassettes

摘要:

裂解作为合成生物学中的常见功能模块单元,在基因线路设计方面具有广泛的运用。裂解主要通过诱导表达噬菌体的裂解基因盒子实现,不过,尚未有关于裂解基因的详细表征。本研究首先利用阿拉伯糖和鼠李糖诱导系统在大肠杆菌(Escherichia coli) Top10表达了5种裂解基因盒子(S₁₀₅, A52G, C51S S76C,LKD, LUZ),通过OD₆₀₀值的测量,表征了在不同生长阶段、诱导剂浓度、质粒拷贝数等条件下,含有不同裂解基因盒子的大肠杆菌菌株Top10的裂解行为。结果表明,虽然5种裂解基因盒子在大肠杆菌内都可以有效引起宿主的裂解,但是裂解行为在不同条件下有较大差异。进一步地,由于诱导系统在不同宿主之间背景表达水平的差异,导致在铜绿假单胞菌(Pseudomonas aeruginosa) PAO1中难以构建可诱导的裂解系统。经过筛选,通过基因组插入的方式,利用鼠李糖诱导系统对裂解基因盒子在铜绿假单胞菌中引起的裂解行为进行表征。结果显示,LUZ和LKD具有较强的使铜绿假单胞菌裂解的能力,而S₁₀₅、A52G、C51S S76C对铜绿假单胞菌的裂解能力较差。最后,利用LUZ和光遗传学工具BphS,通过调整核糖体结合位点(ribosome binding site, RBS)的强度,构建了可在指定表面黏附并裂解的工程菌Q16,该菌株具有应用于表面修饰的巨大潜力。

关键词:裂解基因盒子;诱导系统;合成生物学;光遗传学;表面修饰

Abstract:

Lysis is a common functional module in synthetic biology and is widely used in genetic circuit design. Lysis could be achieved by inducing expression of lysis cassettes originated from phages. However, detailed characterization of lysis cassettes hasn't been reported yet. Here, we first adopted arabinose- and rhamnose-inducible systems to develop inducible expression of five lysis cassettes (S₁₀₅, A52G, C51S S76C, LKD, LUZ) in Escherichia coli Top10. By measuring OD₆₀₀, we characterized the lysis behavior of strains harboring different lysis cassettes. These strains were harvested at different growth stages, induced with different concentrations of chemical inducers, or contained plasmids with different copy numbers. We found that although all five lysis cassettes could induce bacterial lysis in Top10, lysis behaviors differed a lot at various conditions. We further found that due to the difference in background expression levels between strain Top10 and Pseudomonas aeruginosa PAO1, it was hard to construct inducible lysis systems in strain PAO1. The lysis cassette controlled by rhamnose-inducible system was finally inserted into the chromosome of strain PAO1 to construct lysis strains after careful screen. The results indicated that LUZ and LKD were more effective in strain PAO1 than S₁₀₅, A52G and C51S S76C. At last, we constructed an engineered bacteria Q16 using an optogenetic module BphS and the lysis cassette LUZ. The engineered strain was capable of adhering to target surface and achieving light-induced lysis by tuning the strength of ribosome binding sites (RBSs), showing great potential in surface modification.

Key words:lysis cassette;inducible systems;synthetic biology;optogenetics;surface modification