(175d) GT DNA Assembly Standard

The construction of plasmids with versatile biotechnological functions is a routine work in almost all genetic engineering related projects. Researchers are currently using various DNA assembly methods to construct plasmid by assembling multiple DNA parts. Extensive research efforts have been made to develop universal worldwide DNA assembly standard to help researches standardize the practices of plasmid construction and facilitate the sharing of standardized DNA parts in research community. Two DNA assembly standards (BioBrick and BASIC) that rely on the use of standardized DNA parts have been developed, but has not been widely adopted so far. The underlying methods of these two standards are restriction endonuclease-based cloning, which inevitably will leave scars (extra DNA sequences) between two DNA parts, potentially affecting the biological function of DNA parts. The scars can be avoided by using overlapping sequences-based DNA assembly methods (e.g., Golden Gate assembly, Gibson method, CLIVA, In-fusion cloning, Yeast assembler), which have been widely used to assemble multiple DNA parts seamlessly. However, employing overlapping sequences-based methods has sacrificed the use of standardized DNA parts, and requires ordering customized oligonucleotides (oligos), which is costly and time-consuming (at least one day for the delivery of oligos). As a result, there was a need to develop a DNA assembly standard that can overcome all the aforementioned limitations, and can also be compatible with varied types of DNA assembly methods.

Here, we report GT standard (GTS) for plasmid construction under which DNA sequences are defined as two types of standard, reusable parts (fragment and barcode). We developed 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 method, In-fusion cloning, restriction endonuclease-based methods and Yeast in vivo assembly. The oligos used to generate the barcoded fragments can be reused when fragments and barcodes are combined in a different order. Based on 436 plasmids we constructed under GTS, the averaged accuracy of the workflow was 85.9 %. Plasmids constructed under GTS can be easily edited, and/or be further assembled into more complex plasmids modularly by using standard oligos. GTS can also construct a library of plasmids from a set of fragments and barcodes combinatorically, which has been demonstrated to be useful to optimize complex biological systems, such as metabolic pathways in Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae. GTS provides an open and flexible architecture to the whole research community, allowing users to flexibly define sharable barcode sequence with or without biological functions, and to select DNA assembly methods based on their preference and the requirements of plasmid construction.