Like a Rolling Circle : Developing in-situ genotyping of chromosomal barcodes in the DuMPLING method

• Main Author: Svahn, Fabian
• Format: Others
• Language: English
• Published: Uppsala universitet, Molekylär systembiologi 2021
• Subjects: Rolling circle amplification; genotyping; chromosomal barcodes; RCA; DuMPLING method; DuMPLING; barcoded libraries; microfluidics; Biochemistry and Molecular Biology; Biokemi och molekylärbiologi
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-452675

DuMPLING is a newly developed high-throughput method to study singlecellphenotypes in a pooled and barcoded library using a microfluidicchip. The chip enables parallel biophysical measurements of singlecells, after which in-situ genotyping connects the cells to a certainstrain of the library. The method has been previously applied with abarcoded library, where genotyping was performed on barcodes presenton high copy number plasmids. In this project, I apply and developthe Rolling Circle Amplification method to amplify the signal frombarcodes present on the E. coli chromosome. A small librarycontaining three different chromosomal barcodes is investigated. Veryhigh efficiency of signal generation is achieved for the firstbarcode, good efficiency is achieved for the second, and no signal isachieved for the third. Genotyping is also successfully performed ona strain with two different barcodes present on the chromosome. Thegenotyping method described herein can be applied to screen foradditional barcodes that may be incorporated in a larger library thatin turn can be used to ask important biological questions, forexample using the high throughput DuMPLING method.