In conclusion, a stepwise biochemical approach lends itself to the eventual identification of any remaining functions essential for the synthesis of a minimal cell sustained solely by small molecules. Five states of completion present themselves as tractable goals of an MCP. Namely, the identification of
- the genes listed as missing in ,
- any additional genes and organization necessary experimentally for minimal cell synthesis,
- any dispensable genes,
- biochemical parameters and computational models sufficiently detailed to predict the effects of alterations and
- the missing three-dimensional structures of the gene products and their relevant complexes.
It is difficult to predict how long it will take to debug each of the individual biochemical subsystems or to put them all together; so it is important to bear in mind that there are short-term goals (see the Utility section). Intermediate assembly steps could also be pursued while the gaps in RNA modification knowledge () are being filled. For example, the project to assemble a ribosome under physiological conditions could be carried out without the missing 23S rRNA modification enzymes () by substituting in natural 23S rRNA. Similarly, assembly of self-replication in the absence of functional in vitro-synthesized tRNA substrates could be carried out using cellular total tRNA to enable self-replication from substrates (rather than just small molecules) as a major step towards understanding biological self-replication. This would also allow directed evolution of all of the components except the tRNAs in a more flexible manner than is possible in vivo (e.g. for selecting ribosome mutants that incorporate unnatural amino acids more efficiently).
The biochemical subsystems necessary for an MCP are central, old fields that have lost impetus. Completion within a decade will only be possible through a coordinated filling of the key gaps in knowledge by the cutting-edge laboratories scattered around the world in these fields. It will also require stimulation of rate-limiting fields. For example, although rRNAs and tRNAs can constitute more than 70% of the dry weight of a cell, half of the estimated 60–70 RNA modification enzymes of E. coli
and one-fifth of the tRNAs remain to be characterized (Supplementary Tables S5 and S6
), despite the recent completion of about 300 bacterial whole genome sequences. The momentum of genomics and consequent deluge of computed hypotheses cries out for comparable breakthroughs in experimental tests. Synthetic systems biology projects such as an MCP promise such tests with the added bonus of new applications.