A New High Throughput Method Based On Tcc To Detect Interactions Of Escherichia Coil Chromosome

Bacteria, one of earth’s oldest species, have a much simpler cell structure than eukaryotes. Bacteria also have a unique way of organizing their genetic material and regulating gene expression. Although the bacterial cell structure appears raw and relatively simple, recent studies revealed that its nucleoid is complex with multi-hierarchical structure. Bacterial chromosomes are highly condensed by three orders of magnitude to fit within a cell, and form nucleoids with RNA and proteins. Bacterial chromosomes are not cluttered and compressed in the nucleoid, but arrar ged in regular and reproducible fashions.The Escherichia coli genome is composed of a series of closely linked genes (operons) to regulate gene expression. A regulon is composed of a series of operons regulated by the same transcription factor. Although operons in the same regulon may be far in linear distance, they can still interact at the space level. Some studies have shown that dispersed genes can colocalize with DNA loops during gene expression. Bacterial gene expression can be regulated by remote regulatory elements, and a3D network of gene regulation can be observed. The hierarchical structure of bacterial chromosomes does not only enable adaptation of the bacterial genomes to a highly condensed nuclear area, but also to widely influence the gene regulatory processes of bacteria.High-level interactions exist between bacterial chromosomes that form the hierarchical structure of a nucleoid. To study the interactions of E. coli chromosomes and obtain high-throughput interaction data, we developed a new high-throughput genome-wide assay based on the latest tethered conformation capture (TCC) technology with slight modifications. E. coli was treated with1%formaldehyde at the log growth phase, and cells were cross-linked. The cross-linked sample was then broken ultrasonically without preference. The DNA ends of the ultrasonically treated sample were filled, and a biotin label was used to fix all the proteins. The sample could then be ligated at a very low DNA concentration. After cross-linking was reversed, DNA was purified and cloned into a vector for sequencing to verify the feasibility of the entire protocol. Among all the linked fragments, about10%of the cloned fragments were composed of sequences with different loci. After biotinylation of nucleoid-associated proteins in TCC, cross-linking molecules were ligated on a support to avoid random contact between fragments, thereby increasing fidelity of the interacting fragments. Biotinylated proteins as affinity purification tags could significantly improve the efficiency of high-throughput screening. Therefore, we effectively removed cross-linking between free nucleic acid fragments. Second-generation sequencing was then used to acquire high-throughput interaction data of E. coli chromosomes. High-throughput data analysis using bioinformatics tools revealed that E. coli K-12chromosomes in the log growth phase demonstrated high-level interactions, and most of these interactions preferentially occurred near replication initiation sites. The chromatin interaction peak matched amino acid synthesis.We modified the technology of chromosome conformation capture and TCC. After optimization of the experimental conditions, limited cloning sequencing, and analysis of high-throughput sequencing data, we established a high-throughput method for detecting interactions among E. coli chromosomes. This method could be applied to the second generation of high-throughput sequencing platforms. The established method could reveal data on bacterial chromosome interactions, construct a3D model of bacterial chromosome structure, and reveal the secrets of gene regulatory networks.