Assessing the Role of Chance
Ancestral EF-Tu proteins, resurrected previously by Eric Gaucher (7) exhibit a range of thermostability profiles (from 39.1 °C to 73.3 °C). During the first quarter of 2011, we finalized an experimental methodology that allows us to efficiently delete the two copies of EF-Tu genes present in modern E. coli and insert an ancestral EF-Tu gene in the precise chromosomal location of one of the modern genes. We have accomplished this goal and to date we have placed two ancient EF-Tus (the ~500 million years old, with the melting temperature of 39.1 °C and the 1.3 billion years old, with the melting temperature of 60.2 °C) (named “MB” and “Rip” respectively) for their modern counterpart within E.coli that grows at 37°C. (See Figure 1 for the final recombineering outline) Considering the highly adaptive properties of E. coli and EF-Tu, and the essentiality of EF-Tu for cell viability since this protein shuttles aminoacylated-tRNAs to the ribosome, our replacement of modern genes for ancient genes has indeed provided a scenario for rapid adaptive evolution to take within modern E. coli. (See Figure 2 for the effects of these replacements on the bacterial doubling time). Completion of this aim marks the first time ancient genes have been incorporated inside a bacterial genome and the bacteria containing these ancient genes exhibit a lower fitness than the strain hosting the modern form of the gene (Figure 2 & 3).
Once we obtained and verified the bacteria containing ancient gene 317 (MB strain), we prepared this particular construct for evolution, selection and competition. For this purpose, we have utilized a technique called ‘gene gorging’ (8) and engineered a neutral arabinose marker (Ara +/-) gene in order to obtain pink (Ara+) and red (Ara-) variants of the MB Strain. This phenotype allows us to determine the results of competition assays on tetrazolium-arabinose (TA) plates. We performed various experiments (obtaining growth curves, fitness effects) to validate the neutrality of this engineering on the phenotype of MB strain.
We have been evolving 4 flasks of Ara- and 4 flasks of Ara+ MB strains in 9.9 mL minimal glucose media (DM25) at 37 degrees for over 5 months now. Every day, after 24 hours of growth, we transfer 100 mL of each of the evolved culture into new fresh DM25 media. We calculate the fitness values of these evolved lineages every 100 generations (~16 days) by competition with REL606 (wild-type) as well as MB (unevolved) strains. We also measure the growth curves of the evolved lineages and calculate the effect on doubling times. Here, for simplicity, we are reporting the fitness values of MB strains at ‘generation 500’ (Figure 3). (Values obtained by competing 5-replicate population of each MB strain with REL606 (wild type) for 1 to 4 days, data obtained during Betul Arslan’s visit to Rich Lenski’s lab at MSU (supported by a NAI Research Scholarship). Competition data shows the adaptive evolution of MB lineages, which increases the fitness of these strains by 20% in less than 4 months of evolution. In order to understand the underlying genetic causes of fitness increase, we perform gene and genome sequencing to evolved strains. A preliminary sequencing data shows that one of the MB lineages experienced duplication in the promoter region of modern EF-Tu gene around 300 generation, suggesting that a fitness optimization through regulatory region modification. On going work includes understanding how much does this duplication in the promoter region actually increase the ancient EF-Tu expression and whether such optimization did/will affect the evolutionary trajectory taken by this lineage. While so far we have not identified any mutations in the ancient EF-Tu gene in any MB lineage, currently we are applying whole-genome sequencing to 8 MB-generation-500 strains to understand the effect of evolution in the context of the entire organism (at MSU sequencing facilities) (9-10). Such whole-genome sequencing will be performed every 500-generations. Other on-going work includes dissecting the phenotypic properties of evolved MB lineages (both in population and molecular level), as well as preparing the REL606 strain with a ~1.3 billion year old EF-Tu, Rip, for evolution through gene gorging as described above.
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