Holowko Maciej B, Poh Chueh Loo
Department of Biochemistry, Life Sciences Institute, Yong Loo Lin School of Medicine, NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), National University of Singapore, Singapore, Singapore.
Department of Biomedical Engineering, National University Singapore, Singapore, Singapore.
Methods Mol Biol. 2018;1772:445-456. doi: 10.1007/978-1-4939-7795-6_26.
In the process of constructing and characterizing the whole cell biosensor for Vibrio cholerae detection, two main techniques have been employed-DNA assembly using the Gibson isothermal assembly reaction was used for the assembly of the PCRed plasmid fragments (DNA parts), and microplate fluorescence readings were used for bacterial strain characterization. The general workflow can be summed up as: the in silico designed DNA fragments were assembled by isothermal assembly to be later transformed into Escherichia coli that, in turn, was characterized using the microplate reader. As fine-tuning of the sensor design was required, the process was repeated iteratively until the final strain was created with desired characteristics. This chapter describes in detail this workflow for different constructs which finally led to the creation of the first whole cell biosensor in E. coli for V. cholerae detection.
在构建和表征用于霍乱弧菌检测的全细胞生物传感器的过程中，采用了两种主要技术——使用吉布森等温组装反应进行DNA组装，用于组装PCR扩增的质粒片段（DNA部件），并使用微孔板荧光读数对细菌菌株进行表征。一般工作流程可总结为：通过等温组装将计算机设计的DNA片段组装起来，随后转化到大肠杆菌中，再使用微孔板读数仪对其进行表征。由于需要对传感器设计进行微调，该过程反复进行，直到创建出具有所需特性的最终菌株。本章详细描述了针对不同构建体的此工作流程，该流程最终促成了在大肠杆菌中创建首个用于霍乱弧菌检测的全细胞生物传感器。
