Chaudhari Vishalsingh R, Hanson Maureen R
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA.
Synth Biol (Oxf). 2021 Nov 10;6(1):ysab032. doi: 10.1093/synbio/ysab032. eCollection 2021.
With increasing complexity of expression studies and the repertoire of characterized sequences, combinatorial cloning has become a common necessity. Techniques like BioBricks and Golden Gate aim to standardize and speed up the process of cloning large constructs while enabling sharing of resources. The BioBricks format provides a simplified and flexible approach to endless assembly with a compact library and useful intermediates but is a slow process, joining only two parts in a cycle. Golden Gate improves upon the speed with use of Type IIS enzymes and joins several parts in a cycle but requires a larger library of parts and logistical inefficiencies scale up significantly in the multigene format. We present here a method that provides improvement over these techniques by combining their features. By using Type IIS enzymes in a format like BioBricks, we have enabled a faster and efficient assembly with reduced scarring, which performs at a similarly fast pace as Golden Gate, but significantly reduces library size and user input. Additionally, this method enables faster assembly of operon-style constructs, a feature requiring extensive workaround in Golden Gate. Our format allows such inclusions resulting in faster and more efficient assembly.
随着表达研究以及已表征序列库的日益复杂，组合克隆已成为一种普遍需求。诸如BioBricks和金门克隆法等技术旨在使大型构建体的克隆过程标准化并加快其速度，同时实现资源共享。BioBricks格式提供了一种简化且灵活的方法，可通过紧凑的文库和有用的中间体进行无限组装，但这是一个缓慢的过程，每个循环仅连接两个部分。金门克隆法利用IIS型酶提高了速度，每个循环可连接多个部分，但需要更大的元件库，并且在多基因格式中后勤效率低下的问题会显著扩大。我们在此提出一种方法，通过结合这些技术的特点对其进行改进。通过以类似BioBricks的格式使用IIS型酶，我们实现了更快、更高效的组装，且疤痕减少，其执行速度与金门克隆法相似，但显著减小了文库大小并减少了用户输入。此外，该方法能够更快地组装操纵子式构建体，这一特性在金门克隆法中需要大量的变通方法。我们的格式允许此类包含物，从而实现更快、更高效的组装。
