Generating genomes and encoding new abilities into artificial DNA

Generating genomes and encoding new abilities into artificial DNA

Mysterious virus found in Brazil puzzles scientists with its unique genetic makeup This was the motive for the current study, which attempts to rewrite the genome on a large scale, using synonymous codons, in combination with finding the causes of errors in the genome in a systematic manner. The need is for “a broadly applicable high-throughput error diagnosis approach,” whereby the rewriting of the whole genome can be evaluated. The project The researchers tried to synthesize the minimized or stripped-down version of the genome of the freshwater bacterium Caulobacter crescentus -2.0 (C. eth-2.0), a very productive and responsive cell cycle model organism. This bacterium’s transcriptome, ribosomes, and many other measurements are already available as a detailed genome model. The aim was to evolve a no-frills approach to design-test-build technique that will help produce a customized genome that incorporates the essential functions of the cell and little else. The basis of this approach is to build this genome chemically so that the information in the genome could be analyzed for what it tells us about essential genes. The procedure The first step was to extract the DNA parts needed from the native bacterium and join them to form a digital genome, keeping the organization and orientation of genes intact. They also included some marker genes, self-replicating sequences, and a shuttle vector sequence that would allow tight low copy replication in this bacterium at the origin of replication. They ran into a roadblock here when they found that many of the DNA building blocks were not commercially available because of constraints on synthesis. They then decided to test their theory that the use of synonymous codons would make it easier to synthesize the sequence chemically but still keep the biological functions unchanged. Accordingly, they created an optimized design based on the earlier one, with over 10,000 changes in the DNA bases, and successfully removed almost 5700 synthesis constraints. They also wanted to test their theory that chemical synthesis would allow them to find out how accurately the genome is annotated and identify inbuilt functions. They, therefore, included over 100,000 base substitutions within the exons. Thus, they replaced about 56% of all codons by their synonyms. In essence, the amino acid sequence remained the same. Still, all other layers of genetic regulation, including alternative reading frames and other hidden controls within the exons, were reduced to the bare minimum. In fact, from 77% to 95% of these elements were removed. This was to allow the identification of genes which need more than the amino acid sequence to be specified to work properly, by seeing which of the rewritten genes remain functional. The non-functional genes must then be repaired, to eventually yield a completely artificial cell with a full complement of essential functions. The results They found that over 90% of enzyme-encoding genes retained functionality. Over 432 genes, overall, remained functional. About 100 genes became non-functional, probably due to wrong annotations or the presence of unknown genetic controls, or because they don’t code for protein at all. This approach is, therefore, useful to test the accuracy of the annotation of a genome. Four of these genes were repaired in a targeted manner, leading to the identification of an essential element upstream of one of them. In some of them, there were noncoding controls within the exons, implying these genes had been wrongly annotated earlier. The implications The combination of these findings shows that while the scientists have not yet produced a living cell, they have tested and found the approach a promising one to produce designer genes. It promises a highly flexible design capability with low-cost, reliable chemical manufacture. The biggest challenge, as always, is not technical, but the ethical and social issues that can arise if it is misused. Journal reference: Chemical synthesis rewriting of a bacterial genome to achieve design flexibility and biological functionality Jonathan E. Venetz, Luca Del Medico, Alexander Wölfle, Philipp Schächle, Yves Bucher, Donat Appert, Flavia Tschan, Carlos E. Flores-Tinoco, Mariëlle van Kooten, Rym Guennoun, Samuel Deutsch, Matthias Christen, Beat Christen Proceedings of the National Academy of Sciences Apr 2019, 116 (16) 8070-8079; DOI: 10.1073/pnas.1818259116



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