The BioBrick standard makes possible iterated pairwise assembly of cloned parts without any depletion of unique restriction sites. Every part that conforms to the standard is compatible with every other part, thereby fostering a worldwide user community. The assembly methods, however, are labor intensive or inefficient compared to some newer ones so the standard may be falling out of favor. An easier way to assemble BioBricks is described herein. Plasmids encoding BioBrick parts are purified from Escherichia coli cells that express a foreign site-specific DNA methyltransferase, so that each is subsequently protected in vitro from the activity of a particular restriction endonuclease. Each plasmid is double-digested and all resulting restriction fragments are ligated together without gel purification. The ligation products are subsequently double-digested with another pair of restriction endonucleases so only the desired insert-recipient vector construct retains the capacity to transform E. coli. This 4R/2M BioBrick assembly protocol is more efficient and accurate than established workflows including 3A assembly. It is also much easier than gel purification to miniaturize, automate and perform more assembly reactions in parallel. As such, it should streamline DNA assembly for the existing community of BioBrick users, and possibly encourage others to join.

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用于高通量 BioBrick 组装的甲基化酶辅助亚克隆

BioBrick 标准使克隆部件的迭代成对组装成为可能，而不会耗尽任何独特的限制性位点。符合标准的每个部分都与其他所有部分兼容，从而形成了一个全球用户社区。然而，与一些较新的组装方法相比，组装方法是劳动密集型或低效的，因此该标准可能会失宠。本文描述了一种更简单的组装 BioBricks 的方法。编码 BioBrick 部分的质粒是从表达外源位点特异性 DNA 甲基转移酶的大肠杆菌细胞中纯化的，因此每个质粒随后都在体外受到保护，免受特定限制性内切核酸酶的活性影响。每个质粒都经过双重消化，所有得到的限制性片段无需凝胶纯化即可连接在一起。连接产物随后用另一对限制性内切核酸酶进行双重消化，因此只有所需的插入物-受体载体构建体保留转化大肠杆菌的能力。这种 4R/2M BioBrick 组装协议比包括 3A 组装在内的既定工作流程更高效、更准确。小型化、自动化和并行执行更多组装反应也比凝胶纯化容易得多。因此，它应该为现有的 BioBrick 用户社区简化 DNA 组装，并可能鼓励其他人加入。小型化、自动化和并行执行更多组装反应也比凝胶纯化容易得多。因此，它应该为现有的 BioBrick 用户社区简化 DNA 组装，并可能鼓励其他人加入。小型化、自动化和并行执行更多组装反应也比凝胶纯化容易得多。因此，它应该为现有的 BioBrick 用户社区简化 DNA 组装，并可能鼓励其他人加入。