Michael A. Fisher, Kara L. McKinley, Luke H. Bradley, Sara R. Viola, and Michael H. Hecht

1. De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth Michael A. Fisher, Kara L. McKinley, Luke H. Bradley, Sara R. Viola, and Michael H. Hecht PLoS ONE: January 4, 2011 Presented by: Amber Lin & Yuan Zhao

2. "Must the toolkit of life be so restricted?" 20100 possible 100-residue proteins life sustained by only 4,000 in E.coli, ~20,000 in humans challenge of synthetic biology: develop living parts independent of nature generated novel 102-residue 4-helix bundles using binary pattern

3. Strategy that this specific pattern will coincide with structure of natural, folded proteins • Pattern of PNPPNNPPNPPNNP residues Binary Pattern

4. Synthetic proteins rescue auxotrophs transformed synthetic genes into single-gene knockout E.coli strains tested for new viability on minimal medium Four auxotrophs regained viability Verified role of de novo protein versus natural revertant mutation

6. Protein has role in novel bypass pathway? • De Novo protein fails to rescue auxotrophs with deletions at other steps in the biosynthetic pathway Potential Biological Functions of de novo Proteins (1)

7. Altering expression or activity of endogenous protein? • Overexpression of these ‘multicopy supressors’ can rescue deletions • De Novo proteins worked even when multicopy supressors were deleted Potential Biological Functions of de novo Proteins (2)

8. Global metabolic alterations (ie cellular stress response)? • Proteins were folded properly (from circular dichroism spectra) • Failure of de novo mutant to rescue cell indicates function is sequence-specific Potential Biological Functions of de novo Proteins (3)

11. Proteins not selected by evolution or for enzymatic function • Even at 400x higher levels than WT, cells grew slower • Could not detect activity in assay Efficiency of de novo proteins

12. Replacement of 0.1% of E.coli Genome probed ability of de novo sequences to rescue multiple deletions only ~400 E.coli genes are essential and (at least) 0.1% of the genome can be replaced by artificial genes

13. Results  de novo proteins can sustain cell growth 4/27 auxotrophs rescued by de novo proteins de novo proteins operate with different pathway than natural proteins Slow growth, low levels of protein activity

14. Purpose / Significance • Protein selection by structural strategy (binary code) • novel sequences with no similarity to natural proteins can compensate for gene deletion • Synthetic biology still in infancy compared to billions of years of evolution • Flaws • Inclusion of work on replacement of E.Coli genome • failure to determine any pathways, only disprove alternative mechanisms • Relevance of work questionable De Novo Proteins