Evolutionary Stability of a Refactored Phage Genome

Engineered genetic systems are commonly unstable; if propagated, they evolve to reverse or modify engineered elements because the elements impair fitness. A goal of synthetic biology is thus to anticipate and avoid detrimental engineering, but little is yet known about which types of elements cause problems in different contexts. In prior work, 30% of the genome of bacteriophage T7 was “refactored” by the insertion or modification of 65 short sequences that included a useful restriction enzyme site in order to, among other goals, separate genes and their translational initiation regions from each other and from other genetic elements. Although gene sequences and known important regions of regulatory elements were kept intact, the translational efficiency of some genes or element regulatory function might have been compromised. We adapted the refactored phage for rapid growth in two conditions, observing fitness and sequence evolution. As anticipated from the original work, refactoring had major fitness effects in both environments, but most of the fitness costs were recovered upon adaptation. The evolved phages retained 60–70% of the design elements, suggesting they had only minor fitness effects. Approximately half the elements that were lost lie within large deletions commonly observed during adaptation of the wild-type genome. Some elements were lost or modified in parallel between the adaptations without affecting T7 gene sequences, but no obvious correlates can be made. Nevertheless, experimental adaptations are useful for identifying specific synthetic design problems, and we suggest that experimental evolution in conjunction with alternative engineering may also be useful in overcoming those problems.经工程改造的遗传系统通常具有不稳定性：若对其进行传代培养，系统会自发演化以逆转或修饰改造元件，原因在于这类改造元件会损害系统的适合度。因此，合成生物学的核心目标之一是预判并规避有害的工程改造，但目前人们对不同情境下哪些类型的改造元件会引发问题仍知之甚少。在先前的研究中，研究者通过插入或修饰65段短序列（其中包含一个实用的限制性酶切位点 (restriction enzyme site)），对T7噬菌体 (bacteriophage T7) 30%的基因组进行了“重构”，其核心目标之一是将各个基因及其翻译起始区域相互分离，并与其他遗传元件分隔开。尽管基因序列以及调控元件的已知关键区域均得以保留，但部分基因的翻译效率或调控元件的功能可能已受损。本研究将重构后的噬菌体在两种条件下进行适应性传代以实现快速生长，并同步观测其适合度变化与序列演化情况。正如先前研究预期的那样，基因组重构对两种培养条件下的噬菌体适合度均产生了显著影响，但在适应性传代后，大多数适合度成本得以恢复。演化后的噬菌体保留了60%~70%的设计元件，这表明这些元件仅对适合度产生了微弱影响。约半数丢失的设计元件位于野生型基因组适应性传代过程中常见的大片段缺失区域内。部分元件在两次适应性传代中均被丢失或修饰，且未影响T7噬菌体的基因序列，但目前尚未发现这些事件的明确关联特征。尽管如此，实验适应性传代仍可用于识别特定的合成设计缺陷，我们认为，结合实验演化与替代性工程策略，或可有效解决这类合成生物学中的设计问题。