Generation of synthetic genomes
Methods for generating synthetic genomes, for example synthetic genomes having desired properties or viable genomes of reduced size, are disclosed. Also disclosed are synthetic genomes produced by the methods disclosed herein and synthetic cells containing the synthetic genomes disclosed herein.
1. A method for generating a minimal synthetic genome of interest, comprising:
(a) providing a first genome known to sustain viability of a prokaryotic cell; wherein the first genome has a size of no more than 12 megabase pair (Mb);
(b) designing and providing a second genome comprising a reduced number of genes compared to the first genome, wherein designing the second genome comprises modifying the first genome;
(c) dividing each of the first and second genomes into at least three corresponding fragments,
(d) combining at least one fragment of the second genome with fragments of the first genome to generate a third genome having all the three corresponding fragments, further comprising grouping genes related to the same biological process in the at least one fragment of the second genome prior to combining; or grouping genes related to the same biological process in the third genome after combining;
(e) testing the third genome generated in step (d) for sufficiency to sustain viability of the prokaryotic cell; and
(f) identifying the third genome as a minimal synthetic genome of interest if it sustains viability of the prokaryotic cell; otherwise genetically modifying the at least one fragment of the second genome and repeating steps (d)-(f) in one or more iterations until a genome that sustains viability of the prokaryotic cell is obtained in the third genome.
2. The method of claim 1 , wherein the first genome is a Mycoplasma genome.
3. The method of claim 1 , wherein the first genome is a multi-chromosome genome.
4. The method of claim 1 , wherein step (b) further comprises testing the second genome for a set of desired properties selected from the group consisting of: growth rate, ratio of growth rate to genome size, expression level of a gene of interest, ratio of viability to genome size, ratio of viability to expression level of a gene of interest, and ratio of growth rate to expression level of a gene of interest.
5. The method of claim 1 , wherein modifying the first genome is based on the information from literature resources, experimental data, or any combination thereof.
6. The method of claim 5 , wherein the experimental data comprises data related to genes of essential function redundancies (EFR), or data obtained from a mutation study of the first genome, a genome related to the first genome, or any combination thereof.
7. The method of claim 1 , wherein at least one of the at least three fragments is a chromosome of the first or second genome, or a portion of a chromosome of the first or second genome.
8. The method of claim 1 , wherein testing the third genome for sufficiency to sustain viability of a cell comprises introducing the genome into a cell or a cell-like system.
9. The method of claim 1 , wherein modifying at least one fragment of the second genome in step (f) further comprises conducting mutation study of the at least one fragment and modifying the at least one fragment at least partly based on the mutation study.
10. The method of claim 1 , wherein step (c) comprises dividing each of the first and second genomes into between 4 and 20 corresponding fragments.
11. The method of claim 1 , wherein at least one fragment of the second genome is present in an extrachromosomal genetic element.
12. The method of claim 1 , wherein the method generates a plurality of third genomes each having all of the at least three fragments.
13. The method of claim 1 , wherein the combining step comprises chemically synthesizing and assembling the fragments of the first and second genomes to generate the third genome.
14. The method of claim 1 further comprising grouping the genes related to the same biological process as contiguous modules a) in the at least one fragment of the second genome, or b) on the third genome.
15. The method of claim 14 wherein the same biological process is selected from the group consisting of: transport and catabolism, ribosome biogenesis, protein export, DNA repair, transcription, translation, nucleotide biosynthesis, metabolism and salvage, glycolysis, proteolysis, membrane transport, rRNA modification, and tRNA modification.
16. The method of claim 14 wherein step (c) comprises dividing each of the first and second genomes into at least 20 corresponding fragments.
17. The method of claim 14 wherein the same biological process is selected from the group consisting of: transcription, RNA metabolism, translation, protein folding, protein export, a gene encoding RNA, ribosome biogenesis, rRNA modification, and tRNA modification.
18. The method of claim 1 wherein the same biological process is selected from the group consisting of: expression of genome information, preservation of genome information, cell membrane structure and function, and cytosolic metabolism.