DNA末端硫修饰依赖的体外大片段DNA连接法

Assembling long DNA fragments with DNA sulfur modification in vitro

摘要:

【背景】DNA组装技术是基因组合成中的一个关键技术。探索低成本、高效率的基因组合成技术一直是合成生物学的重要研究领域。在某些细菌如变铅青链霉菌中，DNA上有磷硫酰化修饰(简称硫修饰)，而在另一些细菌如天蓝色链霉菌中存在一种含有硫修饰识别结构域(sulfur-binding domain, SBD)的识别蛋白，可以特异性识别DNA上的硫修饰，这启发了我们发展出一种新的DNA组装技术。【目的】探究在DNA末端硫修饰的连接中，T4 DNA连接酶与SBD相融合蛋白和单独的T4 DNA连接酶相比，是否有更高的连接效率。【方法】根据同源重组原理，设计硫修饰引物，扩增硫修饰的DNA片段。构建T4 DNA连接酶与SBD融合蛋白的3种表达载体T4-linker-SBD (Hga)、T4-linker-SBD (Spr)和T4-linker-SBD (Mmo)，表达纯化以上3种融合蛋白。比较3组浓度梯度(2.4、0.24、0.024 mg/mL) T4 DNA连接酶与融合蛋白在2.5 kb和8.0 kb DNA片段连接上的差异。【结果】DNA末端硫修饰的2.5 kb和8.0 kb的两端片段均能扩增，而且3种融合蛋白均能表达。在两段2.5 kb片段的体外连接实验中，每组T4 DNA连接酶与融合蛋白均能连接成5 kb片段；然而在两段8.0 kb片段的体外连接实验中，相同连接时间和相同酶浓度下，仅融合蛋白T4-linker-SBD (Hga)能够将8.0 kb片段连接成16 kb的长片段。【结论】根据两组琼脂糖凝胶电泳的实验结果可知，在两段8.0 kb的DNA长片段连接实验中，T4-linker-SBD (Hga)融合蛋白具有更高的连接效率。本研究方法以较低的成本和比较高的效率为大片段DNA组装提供了一种新的方法和思路。

Abstract:

[Background] Assembling large DNA fragments is crucial in genome synthesis. Exploring inexpensive and efficient DNA assembly technologies has been a research hotspot in synthetic biology. In some bacteria such as Streptomyces lividans, there exists phosphorothioation modification on DNA (also known as DNA sulfur modification), while in some other bacteria such as Streptomyces coelicolor, there is a protein possessing sulfur-binding domain (SBD), which serves as a reader protein to specifically recognize the sulfur modification on DNA. This inspired us to develop a new technology to ligate long DNA fragments. [Objective] In this study, we introduced sulfur modification at the 5′ and 3′ ends of the DNA fragments to be ligated, and employed a fusion protein containing SBD domain and T4 DNA ligase to complete the ligation of long DNA fragments in vitro and compare the different efficiency between 2.5 kb and 8 kb fragments with these two ligases. [Methods] We designed sulfur-modified primers and amplified 2.5 kb and 8 kb DNA fragments with sulfur modification. Additionally, we constructed three fusion protein vectors: T4-linker-SBD (Hga), T4-linker-SBD (Spr), and T4-linker-SBD (Mmo) and expressed them in Escherichia coli and compared the ligation efficiency between T4 DNA ligase and fusion proteins under three different concentration gradients: 2.4, 0.24, 0.024 mg/mL. [Results] We successfully amplified 2.5 kb and 8 kb DNA fragments with sulfur modification and expressed these three fusion proteins. In the 2.5 kb DNA fragments ligation experiment, we found that T4 DNA ligase and fusion protein under the three concentration gradients and in twelve groups all successfully ligated, whereas in the 8 kb DNA fragments ligation experiment, only T4-linker-SBD (Hga) fusion protein successfully ligated two 8 kb fragments into 16 kb fragment. [Conclusion] According to our agarose gel electrophoresis, we concluded that in the 8 kb DNA fragments ligation experiment, T4-linker-SBD (Hga) fusion protein had higher ligation efficiency. Thus our method provide a new way to assemble large DNA fragments with low cost and high efficiency.