A standard for near-scarless plasmid construction using reusable DNA parts

Xiaoqiang Ma,Xiaoyi Cui,Nga Yu Poon,Benjamin Ho,Wenbo Ning,Kang Zhou,Yurou Liu,Hong Liang,Hongyuan Lu

doi:10.1038/s41467-019-11263-0

Abstract

Here we report GT (Guanin/Thymine) standard (GTS) for plasmid construction under which DNA sequences are defined as two types of standard, reusable parts (fragment and barcode). We develop a technology that can efficiently add any two barcodes to two ends of any fragment without leaving scars in most cases. We can assemble up to seven such barcoded fragments into one plasmid by using one of the existing DNA assembly methods, including CLIVA, Gibson assembly, In-fusion cloning, and restriction enzyme-based methods. Plasmids constructed under GTS can be easily edited, and/or be further assembled into more complex plasmids by using standard DNA oligonucleotides (oligos). Based on 436 plasmids we constructed under GTS, the averaged accuracy of the workflow was 85.9%. GTS can also construct a library of plasmids from a set of fragments and barcodes combinatorically, which has been demonstrated to be useful for optimizing metabolic pathways.

Highlights

After the technologies that can assemble multiple DNA parts seamlessly were developed around 2009 (Gibson[8] and Golden Gate[9] methods), research labs swiftly adopted them to speed up projects
GTS has three rules: (1) any DNA sequence longer than 35 nucleotides, starting with G and ending with T can be defined as a fragment; (2) any DNA sequence between 20–80 nt can be defined as a barcode; (3) in plasmid construction any fragment must be placed after a barcode, and any barcode must be placed after a fragment
Promoter can be either fragment or barcode depending on its length (T7 promoter with LacI repressor expression cassette [~1.7 kbp] can be a fragment, while pJ23119 promoter with a length of merely 35 bp can be a barcode)

Summary

Introduction

After the technologies that can assemble multiple DNA parts seamlessly were developed around 2009 (Gibson[8] and Golden Gate[9] methods), research labs swiftly adopted them to speed up projects. They have, mostly used customized parts till to date. We report GT (Guanin/Thymine) DNA assembly standard (GTS) which has met the need, and has the potential to reduce the cost and time of plasmid construction in biotechnological applications if a considerable fraction of research community adopts GTS. GTS enables construction of plasmids in a near-scarless manner by using standard parts and is compatible with CLIVA, Gibson assembly, In-fusion cloning, and RE-based DNA assembly methods. GTS is suitable for constructing plasmid libraries that are very useful in many biotechnological applications, such as metabolic engineering

Methods

Results

Conclusion