Baas-Thomas, M. | bioRxiv Science

bioRxiv2022

Swapped genetic code blocks viral infections and gene transfer

Removing cellular transfer RNAs (tRNAs), making their cognate codons unreadable, creates a genetic firewall that prevents viral replication and horizontal gene transfer. However, numerous viruses and mobile genetic elements encode parts of the translational apparatus, including tRNAs, potentially rendering a genetic-code-based firewall ineffective. In this paper, we show that such horizontally transferred tRNA genes can enable viral replication in Escherichia coli cells despite the genome-wide lack of three codons and the previously essential cognate tRNAs and release factor 1. By repurposing viral tRNAs, we then develop recoded cells bearing an amino-acid-swapped genetic code that reassigns two of the six serine codons to leucine during translation. This amino-acid-swapped genetic code renders cells completely resistant to viral infections by mistranslating viral proteomes and prevents the escape of synthetic genetic information by engineered reliance on serine codons to produce leucine-requiring proteins. Finally, we also repurpose the third free codon to biocontain this virus-resistant host via dependence on an amino acid not found in nature.

synthetic biology↗

bioRxiv2022

Strategies to Identify and Edit Improvements in Synthetic Genome Segments Episomally.

Genome engineering projects often utilize bacterial artificial chromosomes (BACs) to carry multi-kilobase DNA segments on low copy number vectors. However, DNA synthesis and amplification have the potential to impose mutations on the contained DNA segments that can in turn reduce or prevent viability of the final strain. Here, we describe improvements to a multiplex automated genome engineering (MAGE) protocol to improve recombineering frequency and multiplexability. This protocol was applied to recoding an Escherichia coli strain to swap out seven codons to synonymous alternatives genome-wide. Ten 44,402 to 47,179 bp de novo synthesized BAC-contained DNA segments from the recoded strain were unable to complement deletion of the corresponding 33 to 61 wild type genes using a single antibiotic resistance marker. Next-Generation Sequencing was used to identify 1-7 non-recoding mutations in essential genes per segment, and MAGE in turn proved a useful strategy to repair these mutations on the BAC-contained recoded segment when both the recoded and wild type copies of the mutated genes had to exist by necessity during the repair process. This strategy could be adapted to mutation identification and repair for other large-scale genome engineering projects, or for incorporation of small genetic engineering sites for quick protocol adjustments.

synthetic biology↗