Bug mapping and fitness testing of chemically synthesized chromosome X

• DOI: 10.1126/science.aaf4706
• Publication Date: 2017-07-11T17:30:19Z
• Authors: Yi Wu; Bing-Zhi Li; Meng Zhao; Leslie A. Mitchell; Ze-Xiong Xie; Qiu-Hui Lin; Xia Wang; Wen-Hai Xiao; Ying Wang; Xiao Zhou; Hong Liu; Xia Li; Ming-Zhu Ding; Duo Liu; Lu Zhang; Bao-Li Liu; Xiao-Le Wu; Fei-Fei Li; Xiu-Tao Dong; Bin Jia; Wen-Zheng Zhang; Guo-Zhen Jiang; Yue Liu; Xue Bai; Tian-Qing Song; Yan Chen; Si-Jie Zhou; Rui-Ying Zhu; Feng Gao; Zheng Kuang; Xuya Wang; Michael Shen; Kun Yang; Giovanni Stracqua...; Sarah M. Richardson; Yicong Lin; Lihui Wang; Roy Walker; Yisha Luo; Ping-Sheng Ma; Huanming Yang; Yizhi Cai; Junbiao Dai; Joel S. Bader; Jef D. Boeke; Ying-Jin Yuan
• Keywords: Synthetic Biology; Genome Engineering

INTRODUCTION Design and construction of an extensively modified yeast genome is a direct means to interrogate the integrity, comprehensiveness, accuracy knowledge amassed by community date. The international synthetic project (Sc2.0) aims build entirely designer, Saccharomyces cerevisiae genome. designed increase stability genetic flexibility while maintaining cell fitness near that wild type. A major challenge for synthesis lies in identifying eliminating fitness-reducing sequence variants referred as “bugs.” RATIONALE Debugging imperative successfully building fit strain encoding However, it time-consuming laborious replace wild-type genes measure systematically. Sc2.0 PCRTag system, which specifies recoded sequences within open reading frames (ORFs), distinguish from DNA simple polymerase chain reaction (PCR) assay. This system provides opportunity efficiently map bugs related using pooling strategy subsequently correct them. Further, we identify designer sequences, will gaps our gain deeper understanding biology, allowing refinement future design strategies. RESULTS We chemically synthesized chromosome X, synX, be 707,459 base pairs. high-throughput mapping called pooled (PoPM) was developed unexpected during assembly. With this method, genotypes pools colonies with normal or defective are assessed analysis. PoPM method exploits patchwork structure observed majority putative integrants meiotic progeny derived synthetic/wild-type backcross. analysis both specific primers, carried out genomic extracted two clones (normal versus growth defect), can used regions missing pool and, analogously, sections absent growth-defect pool. In way, defect mapped very small overlapping region, subsequent systematic changes region bug. Several were identified corrected, including synonymously essential FIP1 ORF effect introducing loxPsym site unexpectedly altered promoter function nearby gene, ATP2. addition, crossover employed repair massive duplications rearrangements chromosome. debugged synX exhibited high under variety conditions tested competitive strain. CONCLUSION Synthetic X scratch, rigorous, incremental step toward complete whole Thousands modifications revealed extensive efficient PoPM, synthesis, generalizable any watermarked chromosome, several details biology uncovered debugging. Considering numerous gene-associated PCRTags available chromosomes, may represent powerful tool interesting phenotypes mutated strains even relevant genes. It also useful study interactions when phenotype generated alterations more genes, substantially expanding cellular functions. likely phenotype(s) resulting SCRaMbLE system. Characterization debugging mapping. ( Top ) overview X. Bottom Flow diagram (PoPM).