Design and synthesis of a minimal bacterial genome

Abstract

We used whole-genome design and complete chemical synthesis to minimize the 1079-kilobase pair synthetic genome of Mycoplasma mycoides JCVI-syn1.0. An initial design, based on collective knowledge of molecular biology combined with limited transposon mutagenesis data, failed to produce a viable cell. Improved transposon mutagenesis methods revealed a class of quasi-essential genes that are needed for robust growth, explaining the failure of our initial design. Three cycles of design, synthesis, and testing, with retention of quasi-essential genes, produced JCVI-syn3.0 (531 kilobase pairs, 473 genes), which has a genome smaller than that of any autonomously replicating cell found in nature. JCVI-syn3.0 retains almost all genes involved in the synthesis and processing of macromolecules. Unexpectedly, it also contains 149 genes with unknown biological functions. JCVI-syn3.0 is a versatile platform for investigating the core functions of life and for exploring whole-genome design.

Fabrication of nanocomposites and hybrid materials using microbial biotemplates

“…JCVIsyn3.0 retained almost all genes involved in synthesis and processing of macromolecules. However, it also contains 149 genes with unknown biological functions [71]. At present, synthetic biology is enormously challenging, but surely, a promising discipline to creating truly artificial life de novo Fig.…”

Bespoke design of whole‐cell microbial machines

“…The minimal genome developed recently by the J. Craig Venter Institute (JCVI; (Hutchison et al ., 2016) is the smallest known genome capable of sustaining self‐replication of a free‐living organism – albeit one that grows relatively slowly and that requires fairly complex nutritional support. Significant work is required to develop an industrially useful chassis cell using this technology, including improved growth rate and the ability to grow well under stresses typical of an industrial bioprocess (Vickers, 2016).…”

“…While writing DNA has notably lagged behind reading DNA, we are now at the stage where, with a reasonable amount of resources and infrastructure, one can write entire microbial genomes from templates. However, of the 473 genes encoded on the minimal genome, the function of 149 is currently unknown (Hutchison et al ., 2016) – indicating that we still have some way to go to achieve the sufficiently detailed understanding of cellular requirements that would enable true greenfield design. Notwithstanding this, it is fair to say that we are starting to move towards the point where we can seriously consider the ground‐up construction of chassis cells.…”

Transient Expression of Plasmodium berghei MSP8 and HAP2 in the Marine Protozoan Parasite Perkinsus marinus

“…The difference in GC content between P. marinus and P. berghei genome (47.4% vs. 23.7%) (Nikbakht et al, 2014) did not hinder the expression of the selected P. berghei genes in P. marinus. Nevertheless, gene synthesis of genes is currently advantageous due to low cost and speed (Gibson et al, 2008;Hutchison et al, 2016), and future attempts to express Plasmodium spp. genes in P. marinus will benefit from codon optimization by synthesis of the genes of interest.…”