Background: Genomes encode for organisms and thus genome synthesis implies the possibility of organismal synthesis, including the synthesis of organisms without constraint to lineage. Current genome-scale engineering projects are focused on minimization, refactoring, or recoding within the context of existing natural lineages. Minicells arise naturally as anucleate cells that are devoid of heritable genetic material but are capable of gene expression. Thus, minicells may serve as a useful starting point for developing lineage-agnostic organisms encoded by newly-designed synthetic genomes. However, the composition and expression capacity of minicells is fixed at the time of their formation. The possibility of reestablishing cellular growth and division starting from minicells and entirely heterologous synthetic genomes is unknown. Results: We observed expression and segregation of functional proteins among mixed populations of reproducing cells and so-derived anucleate minicells via fluorescence microscopy. By adapting and integrating established methods of preparation and purification we were able to isolate minicells from a growing population of progenitor cells with a purity of at least 500 minicells per progenitor. We then used heterologous expression of plasmid-encoded green fluorescent protein to estimate the absolute expression capacity of minicells. We found that minicells can support the formation of 4.9 +/- 4.6 1E8 peptide bonds prior to exhausting their initial intrinsic expression capacity. Conclusions: Minicells can be produced in large numbers with high purity and can also harbor and express engineered plasmids. The observed variation in minicell gene expression capacity suggests that about 13% of gene-expressing minicells can support the formation of more than one-billion peptide bonds, an amount sufficient to replicate known prokaryotic proteomes. Stated differently, while most minicells would require a sophisticated genetic boot program to first increase minicell-specific expression capacity sufficient to instantiate newly reproducing lineages, a subset of minicells may be able to directly support whole genome, lineage-agnostic organism engineering.

中文翻译：

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小型细胞可作为与工程谱系无关的生物的潜在底盘

背景：基因组编码生物，因此基因组合成暗示了生物合成的可能性，包括不受谱系限制的生物合成。当前的基因组规模的工程项目致力于在现有自然谱系的背景下进行最小化，重构或重新编码。小细胞自然而然地成为无核的遗传细胞，但能够进行基因表达。因此，小细胞可以作为开发由新设计的合成基因组编码的与谱系无关的生物的有用起点。然而，小细胞的组成和表达能力在它们形成时是固定的。从小细胞和完全异源的合成基因组开始重建细胞生长和分裂的可能性尚不清楚。结果：我们通过荧光显微镜观察到生殖细胞和如此衍生的无核小细胞的混合种群中功能蛋白的表达和分离。通过适应和整合已建立的制备和纯化方法，我们能够从不断增长的祖细胞群中分离出小细胞，每个祖细胞的纯度至少为500个小细胞。然后，我们使用质粒编码的绿色荧光蛋白的异源表达来估计小细胞的绝对表达能力。我们发现，在耗尽其初始内在表达能力之前，小细胞可以支持4.9 +/- 4.6 1E8肽键的形成。结论：小细胞可以大量生产，纯度高，也可以携带和表达工程质粒。观察到的小细胞基因表达能力的变化表明，约有13％的基因表达小细胞可以支持形成十亿个以上的肽键，足以复制已知的原核蛋白质组。换句话说，虽然大多数小细胞需要一个复杂的基因启动程序来首先增加足以实例化新近复制的谱系的小细胞特异性表达能力，但一部分小细胞可能能够直接支持整个基因组，与谱系无关的生物工程。