The genome of the laboratory strain E. coli K12 encodes for approximately 4300 proteins, but only a small fraction of their enantiomeric counterparts have been reported. Many of these proteins bear intrinsic synthetic challenges, because of their size, post-translational modifications and folding (see section 3). Construction of the necessary mirror-image oligonucleotides is also a major challenge. Whilst the synthesis of relatively short oligonucleotides is possible from L-xylose or L-arabinose, the resulting oligonucleotides suffer from lower purity. With the advent of the high fidelity D-Pfu capable of assembling complex genes, one might argue that this challenge is within reach. Given the significant efforts, it is also expected that a mirror-image ribosome from bacteria will soon be reported. In addition, mirrorimage tRNAs, tRNA synthetases, and translation factors will need to be prepared to enable the translation of L-mRNA to Dpolypeptides. When made available, the mirror-image translation system will be game-changing in the landscape of enantiomeric protein synthesis. Finally, pursuit of a truly self-replicating system will require an approach of devising a minimal cell and assembly of each essential component in mirror-image form.