大肠杆菌比酿酒酵母富集DNA能力强，是人工染色体合成与组装的良好底盘，但缺少超大容量载体是大肠杆菌中组装大片段DNA的限制因素之一。霍乱弧菌II号染色体（ChrIIvc）复制区能容载1.07 Mb的基因组，是开发大肠杆菌超大容量载体的理想选择。申请者拟基于ChrIIvc复制区在大肠杆菌中组装超大线形人工染色体，主要研究：1）考察ChrIIvc复制区各元件与线形人工染色体的稳定性、拷贝数的关系，确定线形人工染色体所必需的关键元件、模块；2）基于团队前期合成的酿酒酵母V和X号染色体DNA材料和大肠杆菌循环迭代组装方法，通过探讨复制区的位置、数目因素，验证线形人工染色体的容载能力，组装超大线形人工染色体；3) 通过测定DNA复制周期、复制调控节点，研究超大线形人工染色体与大肠杆菌基因组复制调控的互作关系。 本研究为大肠杆菌中人工染色体的合成与组装提供一个超大容量线形染色体载体。

Escherichia coli is more capable of enriching DNA than Saccharomyces cerevisiae and it is an good bottom cell for artificial chromosome synthesis. But the lack of large capacity vector limits the synthesis of large artificial chromosomes in Escherichia coli. The replicon of Vibrio cholerae chromosome II (ChrIIvc) has the capacity of 1.07 Mb. It is an ideal choice for the development of super-large vector of Escherichia coli. The applicant aims to assemble super-large linear artificial chromosome in Escherichia coli based on ChrIIvc replicon. The main research will be: 1) By investigating the relationship between replicon parts of ChrIIvc and the stability and copy numbers of the linear artificial chromosome, determine the key parts and modules necessary for linear artificial chromosome design. 2) Based on the earlier synthesized DNA materials of Saccharomyces cerevisiae chromosome V and X by our team, and on the iterative assembly method in Escherichia coli, the capacity of linear artificial chromosome is verified by exploring the location and number of the replicon. And then to assembly super-large linear artificial chromosomes; 3) By studying on the relationship of replication regulation between the super-large linear artificial chromosome and the Escherichia coli chromosome with measuring the DNA replication cycle and the replication regulation mode. The study will provide a large capacity of linear chromosome vector for the synthesis and assembly of artificial chromosomes in in Escherichia coli .