Tuesday, June 27, 2017

Bacterial genome recoding with the BioXp™ System and Gibson Assembly®


Genome-modification technologies, including creating minimal synthetic genomes, gene editing through directed nucleases, such as CRISPR-Cas9 and genome recoding, offer targeted rational design and engineering at the whole organism level. Genome recoding (systematically altering targeted sense codons throughout a genome or defined genome section) is an important emerging synthetic biology field with many potential applications. Objectives of genome recoding include protein engineering with nonstandard amino acids; retooling organisms to generate bio-based pharmaceuticals, nutritionals, or vaccines; redesigning organisms with novel functions; generation of biocontainment methodologies; and creating synthetic organisms to serve as models for elucidating the basic principles of life.

Large-scale genome recoding

Recently, the Silver laboratory at Harvard University published a report describing the largest cumulative bacterial recoding project to date, in which 5% of the Salmonella genome was rewritten. Specifically, within two genomic regions spanning a total of 200 kb, every TTA codon was rewritten to CTA and every TTG codon was rewritten to CTG. These nucleotide changes were silent since all code for leucine. This proof-of-concept report, including the development of a novel method (SIRCAS) to achieve this large-scale feat, sets the stage for Silver’s laboratory to remove every TTA and TTG throughout the entire Salmonella genome. A genetically recoded organism (GRO) devoid of TTA and TTG, and their cognate tRNAs, would not have the ability to translate the missing codons and would be a genetic isolate, unable to properly translate DNA acquired from other cells, viruses, or plasmids. Potential applications of GRO bacteria include creating more effective live vaccines and acting as a living diagnostic and therapeutic agent in the human gut.

Achieving large-scale recoding

To accomplish such a sizeable undertaking, Pam Silver and colleagues purchased 2 to 4 kb synthetic DNA fragments containing the nucleotide changes from commercial vendors, including SGI-DNA. However, a portion of the fragments could not be synthesized commercially. A small fraction of these regions contained highly repetitive DNA which were PCR-amplified and mutagenized from genomic DNA. The remaining fragments were built in-house in the Silver laboratory using the SGI-DNA BioXp3200 instrument. Unlike commercially synthesized DNA from a now defunct commercial vendor, DNA built on the BioXp system did not have any forbidden restriction enzyme site design constraints and did not contain any extraneous flanking sequences requiring downstream removal. Additionally, DNA built on the BioXp instrument could be conveniently built on site.

After obtaining all requisite DNA constructs spanning the target regions, the 1 to 4 kb DNA fragments were assembled into 10 to 25 kb segments using the Gibson Assembly® method. The 10 to 25 kb segments were then transformed into yeast spheroplasts. Successful assemblies were identified and used for rolling circle amplification and subsequent integration into Salmonella using a new method Silver's group developed called SIRCAS (step-wise integration of rolling circle amplified segments). This project resulted in recoding over 1500 leucine codons within the Salmonella genome and is the first non-E. coli bacterial recoding project as well as the largest bacterial recoding project reported to-date.

This study demonstrates a novel way to achieve large-scale bacterial genome recoding using SGI-DNA BioXp™ System and Gibson Assembly® technologies. Please click here to read the full report.



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