1. Presented by: Amber Lin & Yuan Zhao
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. Binary Pattern
Strategy that this specific pattern will coincide with structure of natural, folded proteins
Pattern of PNPPNNPPNPPNNP residues

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

7. Potential Biological Functions of de novo Proteins (2)
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

8. Potential Biological Functions of de novo Proteins (3)
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

11. Efficiency of de novo proteins
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

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. De Novo Proteins Purpose / Significance Flaws
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