Lineage tracing via associative chromosome/plasmid barcoding with siBar

We have developed siBar, an experimental framework to simultaneously barcode chromosomes and plasmids at the lineage-specific level in E. coli. The siBar rationale is to establish permanent chromosome/plasmid association by introducing unique pairs of chromosomal/plasmid barcodes. This is done by integrating a construct containing a linearized plasmid and two barcodes into the chromosome, and further excising the plasmid and one barcode in such a way that the other barcode remains on the chromosome.Before plasmid excision, the chromosomal and plasmid barcodes are in close promixity, and are covered in one amplicon (D2_P1) during sequencing library prepatation. After plasmid excision, the chromosomal and plasmid barcode regions are amplified in two amplicons, denoted D2_P2 and D2_P3, respectively. The cell culture after plasmid excision was propogated in M63 broth with galactose as the sole carbon source, and LB broth. Both lines were serially transferred with increasing tetracycline (Tc) concentrations. In the M63 line, 10 ug/mL Tc was first added, then 30 ug/mL Tc. In the LB line, Tc was used in the order of 10, 30, 50, 100 ug/mL.The M63 line sample under 10 ug/mL Tc was used to amplify the chromosomal and plasmid barcode regions, denoted D2_P4 and D2_P5, respectively. The LB line sample under 100 ug/mL Tc was used to amplify the chromosomal and plasmid barcode regions, denoted D2_P6 and D2_P7, respectively.