Measurements and analyses of transposable element activity inside living cells

Transposable element;insertion sequence;IS608;transposase;TnpA;retrotransposon;LINE-1;L1;Moran model;mutation;time-lapse microscopy;fluorescence microscopy;synthetic biology;genome editing

Kim, Hyuneil

关键词: Transposable element;    insertion sequence;    IS608;    transposase;    TnpA;    retrotransposon;    LINE-1;    L1;    Moran model;    mutation;    time-lapse microscopy;    fluorescence microscopy;    synthetic biology;    genome editing;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/99445/KIM-DISSERTATION-2017.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Transposable elements (TEs) are DNA elements that move into different places in the DNA. Through their activity, they can restructure genomes and play crucial roles in evolution, development, and genetic disease. However, characterization of their detailed in vivo dynamics has been limited by a lack of direct observational methods. Here, we present novel methods that quantify biophysical characteristics of TEs in unprecedented detail.For the first study, we modify the bacterial transposable element IS608 to quantify its activity in single cells via fluorescence microscopy. The system can reveal single events, cell-to-cell variations, and temporal and environmental variabilities in real time and individual living cells. With this system, we characterize the relationship of the level of transposase protein with TE activity for different orientations of the TE in the genome. We also perform real-time activity detection and find that the activity is highly variable depending on the growth phase, local environment, and growth history of host cells.Secondly, we copy from the human genome a long interspersed nuclear element, LINE-1, one of the most prevalent and active transposable elements in humans. We introduce LINE-1 into the bacteria Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) and demonstrate that it is active in the bacteria and successfully integrates into the bacterial host genomes. The LINE-1 activity decreases the bulk growth rate of bacteria exponentially in response to its increasing expression. Our work suggests that E. coli can be a simple and useful model system to investigate the biophysical properties of LINE-1 element dynamics and their effects on host cells.In the last chapter, we present the technical development of a novel method for versatile and precise genome editing of E. coli, based on the Landing Pad intermediate method suited for the integration of large size DNA fragments at arbitrary locations in E. coli chromosome. Various genome modifications were made to show the power of this method, which include antibiotic-free selection methods, exact integration of long sequences (~6.5kbp) to any given target location, scar-less deletions, and gene fusion to native genes in situ.