Background The comparatively small genome, well elucidated functional genomics and rapid life cycle confer T7 bacteriophage with great advantages for bio-application. Genetic manipulation of T7 genome plays a key role in T7 related applications. As one of the important aspects in T7 phage genetic modification, gene knock-in refers to two main approaches including direct genetic manipulation in vitro and recombineering. Neither of these available methods are efficient enough to support the development of innovative applications capitalizing on T7 bio-system and thus there is room for novel strategies that address this issue. Integration mediated by the ΦC31 integrase is one of the most robust site-specific recombination systems. ΦC31 integrases with enhanced activity and specificity have been developed such that it is ideal to effectuate exogenous DNA knock-in of T7 phage with advanced ΦC31 integrase. Methods Plasmid construction was conducted by routine molecular cloning technology. The engineered T7 bacteriophages were constructed through homologous recombination with corresponding plasmids and the functional T7 phage was designated as T7∆G10G11-attB. In the integration reaction, hosts with both executive plasmids (pEXM4) and donor plasmids (pMCBK) were lysed by T7∆G10G11-attB. Progenies of T7 phages that integrated with pMCBK were isolated in restrict hosts and validated by sequencing. T7∆G10G11-attB capacity limit was explored by another integration reactions with donor plasmids that contain exogenous DNA of various lengths. Results T7∆G10G11-attB exhibits abortive growth in restrictive hosts, and a bacterial attachment site recognized by ΦC31 integrase (attB) was confirmed to be present in the T7∆G10G11-attB genome via sequencing. The integration reaction demonstrated that plasmids containing the corresponding phage attachment site (attP) could be integrated into the T7∆G10G11-attB genome. The candidate recombinant phage was isolated and validated to have integrated exogenous DNA. The maximum capacity of T7∆G10G11-attB was explored, and it's found that insertion of exogenous DNA sequences longer than 2 kbp long can be accommodated stably. Conclusion We advanced and established a novel approach for gene knock-in into the T7 genome using ΦC31 integrase.

中文翻译：

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T7噬菌体基因组整合外源DNA的新方法。

背景 相对较小的基因组、明确的功能基因组学和快速的生命周期赋予了T7噬菌体在生物应用方面的巨大优势。T7 基因组的遗传操作在 T7 相关应用中起着关键作用。作为T7噬菌体基因改造的重要方面之一，基因敲入是指体外直接基因操作和重组工程两种主要途径。这些可用方法都不足以支持利用 T7 生物系统开发创新应用程序，因此存在解决该问题的新策略的空间。由 ΦC31 整合酶介导的整合是最强大的位点特异性重组系统之一。已经开发出具有增强的活性和特异性的 ΦC31 整合酶，因此它是用先进的 ΦC31 整合酶实现 T7 噬菌体的外源 DNA 敲入的理想选择。方法采用常规分子克隆技术构建质粒。通过与相应质粒同源重组构建工程化的T7噬菌体，功能性T7噬菌体命名为T7ΔG10G11-attB。在整合反应中，具有执行质粒 (pEXM4) 和供体质粒 (pMCBK) 的宿主被 T7ΔG10G11-attB 裂解。在限制性宿主中分离出与 pMCBK 整合的 T7 噬菌体后代，并通过测序验证。T7ΔG10G11-attB 容量限制是通过与含有各种长度的外源 DNA 的供体质粒的另一个整合反应来探索的。结果 T7ΔG10G11-attB 在限制性宿主中表现出中止生长，并且通过测序证实了被ΦC31整合酶（attB）识别的细菌附着位点存在于T7ΔG10G11-attB基因组中。整合反应表明含有相应噬菌体附着位点 (attP) 的质粒可以整合到 T7ΔG10G11-attB 基因组中。候选重组噬菌体被分离并验证具有整合的外源DNA。探索了T7ΔG10G11-attB的最大容量，发现可以稳定地容纳长度超过2 kbp的外源DNA序列的插入。结论 我们提出并建立了一种使用 ΦC31 整合酶将基因敲入 T7 基因组的新方法。并且通过测序证实了被ΦC31整合酶（attB）识别的细菌附着位点存在于T7ΔG10G11-attB基因组中。整合反应表明含有相应噬菌体附着位点 (attP) 的质粒可以整合到 T7ΔG10G11-attB 基因组中。候选重组噬菌体被分离并验证具有整合的外源DNA。探索了T7ΔG10G11-attB的最大容量，发现可以稳定地容纳长度超过2 kbp的外源DNA序列的插入。结论 我们提出并建立了一种使用 ΦC31 整合酶将基因敲入 T7 基因组的新方法。并且通过测序证实了被ΦC31整合酶（attB）识别的细菌附着位点存在于T7ΔG10G11-attB基因组中。整合反应表明含有相应噬菌体附着位点 (attP) 的质粒可以整合到 T7ΔG10G11-attB 基因组中。候选重组噬菌体被分离并验证具有整合的外源DNA。探索了T7ΔG10G11-attB的最大容量，发现可以稳定地容纳长度超过2 kbp的外源DNA序列的插入。结论 我们提出并建立了一种使用 ΦC31 整合酶将基因敲入 T7 基因组的新方法。整合反应表明含有相应噬菌体附着位点 (attP) 的质粒可以整合到 T7ΔG10G11-attB 基因组中。候选重组噬菌体被分离并验证具有整合的外源DNA。探索了T7ΔG10G11-attB的最大容量，发现可以稳定地容纳长度超过2 kbp的外源DNA序列的插入。结论 我们提出并建立了一种使用 ΦC31 整合酶将基因敲入 T7 基因组的新方法。整合反应表明含有相应噬菌体附着位点 (attP) 的质粒可以整合到 T7ΔG10G11-attB 基因组中。候选重组噬菌体被分离并验证具有整合的外源DNA。探索了T7ΔG10G11-attB的最大容量，发现可以稳定地容纳长度超过2 kbp的外源DNA序列的插入。结论 我们提出并建立了一种使用 ΦC31 整合酶将基因敲入 T7 基因组的新方法。