A Synthetic Genetic Circuit Enables Precise Quantification of Direct Repeat Deletion in Bacteria

    Repeated sequences exist extensively in the genomes of prokaryotic and eukaryotic cells and the incidence of quantitative repeat deletion is an important means to study DNA rearrangement.
Traditional quantitative methods based on resistance genes need to introduce antibiotic screening, which will not only bring high false positive rate, but also may affect the physiological process of host cells.
At the same time, the quantitative method based on the resistance gene is limited by the length of the coding sequence of the resistance gene, which directly affects the quantitative study of the repeat sequence length in the quantification of the deletion rate of the repeat sequence.

    Aiming at the above problems and the deficiencies of the original method, the Jinfan team designed a set of gene circuits to directly associate DNA rearrangement events with the expression of different colored fluorescent proteins, and then directly read out the rearrangement events occurring in the bacterial population through high-throughput single bacteria data collection and data analysis.
    Experimental results show that in pseudomonas aeruginosa, removing rate and repeat sequence homology arm length into type 'S' relations, and long in the same arm, repetitive sequence removed rate showed a rec a protein dependency, and in a short homologous arm, repetitive sequence deletion rate showed rec a protein of dependence, the addition of the antibiotic ciprofloxacin can significantly increase the rate of repeat sequences to delete, but does not affect repetitive sequence deletion ratio's relationship with the same arm length.
It was also found that the recombinant genes uvrD and radA did significantly increase the rate of repeat deletion and were associated with RecA proteins.
    The fluorescent protein-based synthetic gene loop system provides a new method for the quantification of repeat deletion rate and a new idea for the study of DNA rearrangement.

    The results in the near future to 'A Synthetic based Circuit Enables Precise Quantification of Direct Repeat Deletion in Bacteria' about the international academic journal ACS Synthetic Biology (https://doi.org/10.1021/acssynbio.9b00256).
    This research is the result of cooperation between Yang Guang's team and Jinfan Team of Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences.
    Huang Yajia, a postdoctoral fellow at the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, is the first author of the paper. Huazhong University of Science and Technology is the first unit.
The research was supported by the Ministry of Science and Technology's "Special Project for Synthetic Biology" and the National Natural Science Foundation of China.