Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
Biotechnol Bioeng. 2021 Oct;118(10):3962-3972. doi: 10.1002/bit.27872. Epub 2021 Jul 6.
Agrobacterium tumefaciens is a soil-borne bacterium that is known for its DNA delivery ability and widely exploited for plant transformation. Despite continued interest in improving the utility of the organism, the lack of well-characterized engineering tools limits the realization of its full potential. Here, we present a synthetic biology toolkit that enables precise and effective control of gene expression in A. tumefaciens. We constructed and characterized six inducible expression systems. Then, we optimized the one regulated by cumic acid through amplifier introduction and promoter engineering and evaluated its 15 cognate promoters. To establish fine-tunability, we constructed a series of spacers and a promoter library to systematically modulate both translational and transcriptional rates. We finally demonstrated the application of the tools by co-expressing genes with altered expression levels using a single signal. This study provides precise expression tools for A. tumefaciens, facilitating rational engineering of the bacterium for advanced plant biotechnological applications.
根癌农杆菌是一种土壤细菌，以其 DNA 递送能力而闻名，被广泛用于植物转化。尽管人们一直有兴趣提高该生物体的实用性，但缺乏特征明确的工程工具限制了其潜力的充分实现。在这里，我们提出了一个合成生物学工具包，可实现根癌农杆菌中基因表达的精确和有效控制。我们构建并表征了六个诱导表达系统。然后，我们通过引入放大器和启动子工程优化了受累积酸调控的表达系统，并对其 15 个同源启动子进行了评估。为了建立精细调控能力，我们构建了一系列间隔子和启动子文库，以系统地调节翻译和转录速率。最后，我们通过使用单个信号表达具有改变表达水平的基因来证明工具的应用。这项研究为根癌农杆菌提供了精确的表达工具，有助于对该细菌进行理性工程设计，以应用于先进的植物生物技术。
