Multiplexed in vivo assembly of long and complex DNA

项目总结/摘要 长而复杂的DNA的高通量、低成本合成可以通过促进以前无法获得的基因和遗传途径的生产和表征，开辟合成生物学、药物开发和基因组学的新前沿。微阵列打印技术提供了高通量和低成本合成几乎任何寡核苷酸长达300个碱基，而新技术，如酶合成，有可能从头合成序列高达2kb。为了构建2- 5 kb DNA块，可以使用一套体外技术组装寡核苷酸，包括聚合酶循环组装、吉布森组装和金门组装。然而，应用体外技术组装长于约2- 5 kb的DNA需要相当长的动手时间，难以多重化和规模化，对于构建复杂DNA（例如极端GC含量、均聚物、重复序列、DNA结构）是不可靠的，并且对于长于约7 kb的DNA是极其具有挑战性的。此外，目前筛选序列完美克隆的组装体的方法是昂贵的，并且需要相当多的动手时间或复杂的机器人技术。为了克服这些障碍，我们开发了一种新的，低成本的，高度多重的体内方法来组装长而复杂的DNA，并测序验证克隆。在这种方法中，DNA块的阵列被引入质粒和细菌中，并且这些阵列通过细菌交配顺序地缝合在一起。组装后，通过另一轮细菌交配对细菌阵列进行条形码化，从而能够从合并的克隆中进行大规模并行DNA分离和测序文库制备。与现有的体外克隆相比，我们用于将寡核苷酸或DNA块拼接在一起的方法便宜高达100倍，并且需要的动手时间比现有技术少100倍，而总时间没有增加。重要的是，体内组装可以大规模组装至少15 kb的DNA，并且包含许多复杂DNA区域。在这里，我们建议通过i）构建用于体内DNA组装和序列验证的高通量生产管道，以及ii）构建有助于跟踪和预测体内DNA组装错误模式的序列分析管道和数据库，来强化，扩展和商业化这项技术。这些目标将使BacStitch最初能够提供定制服务，并最终提供标准化产品，以满足长期和复杂DNA的未满足需求。

PROJECT SUMMARY/ ABSTRACT High-throughput, low-cost synthesis of long and complex DNA could open up new frontiers in synthetic biology, drug development, and genomics by facilitating the production and characterization of previously inaccessible genes and genetic pathways. Microarray printing technologies provide for high-throughput and low-cost synthesis of almost any oligonucleotide up to 300 bases long, while new technologies, such as enzymatic synthesis, have the potential to synthesize sequences de novo up to 2kb. To construct 2-5kb DNA blocks, oligonucleotides can be assembled using a suite of in vitro technologies, including polymerase cycling assembly, Gibson assembly, and Golden Gate assembly. However, application of in vitro technologies to assemble DNA longer than ~2-5kb requires considerable hands-on time, is difficult to multiplex and scale, is unreliable for construction of complex DNA (e.g. extreme GC content, homopolymers, repeats, DNA structure), and is extremely challenging for DNA longer than ~7kb. In addition, current methods to screen assemblies for sequence perfect clones are expensive and require considerable hands-on time or complex robotics. To overcome these hurdles, we have developed a novel, low-cost, highly multiplexed in vivo method to assemble long and complex DNA, and to sequence verify clones. In this method, arrays of DNA blocks are introduced into plasmids and bacteria, and these arrays are stitched together sequentially by bacterial mating. Following assembly, bacterial arrays are barcoded via another round of bacterial mating, enabling massively parallel DNA isolation and sequencing library preps from pooled clones. Compared to existing in vitro cloning our approach for stitching either oligos or DNA blocks together is up to 1 00X cheaper and requires up to 100X less hands-on time than current technologies, with no increase in total time. Importantly, in vivo assembly can assemble, at scale, DNA that is at least 15kb and that contains many regions of complex DNA. Here, we propose to harden, scale, and commercialize this technology by i) building a high-throughput production pipeline for in vivo DNA assembly and sequence validation, and ii) building a sequence analysis pipeline and database that will aid in both tracking and predicting error modes of in vivo DNA assembly. These aims will enable BacStitch to initially provide custom services and eventually standardized products that fulfill an unmet need for long and complex DNA.