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1.
Figure 5

Figure 5. Rescue of a quadruple knockout E. coli by co-expression of 4 de novo proteins.. From: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth.

(A) Schematic of knockout rescue. Top: Wild-type E. coli transformed with empty vector forms colonies on M9/glucose minimal media. Middle: QUAD E. coli transformed with empty vector does not form colonies on minimal media. Bottom: QUAD transformed with a vector co-expressing four de novo sequences forms colonies on minimal media. (B) Colonies are not observed when QUAD is transformed with empty vector (top panel), but are observed when transformed with a vector expressing four artificial genes (bottom panel). Scale bar = 1 mm.

Michael A. Fisher, et al. PLoS One. 2011;6(1):e15364.
2.
Figure 3

Figure 3. Designed amino acid sequences that enable growth of E. coli auxotrophs on selective media.. From: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth.

Names of the novel (Syn) proteins and the auxotroph rescued are listed at left. The novel sequences follow the designed binary pattern of polar (red) and nonpolar (yellow) residues. The top line shows the template designed to encode four amphiphilic alpha-helices. Combinatorial positions are shown as empty circles (polar residues), filled circles (nonpolar residues), or hyphens (turn residues). Sequences were submitted to the European Nucleotide Archive and assigned accession numbers FR718891 - FR718908.

Michael A. Fisher, et al. PLoS One. 2011;6(1):e15364.
3.
Figure 4

Figure 4. Growth of auxotrophic strains of E. coli in selective liquid media.. From: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth.

In each panel, the bottom curve shows that the negative control expressing LacZ (open circles) does not support growth. In contrast, most of the positive controls (black circles) expressing the natural E. coli proteins grow well. (Fes is an exception because overexpression of E. coli Fes is toxic.) The designed proteins enable growth that is well above background. (A) ΔserB: LacZ, open circles; SerB, closed circles; Syn-SerB-1, closed triangles; Syn-SerB-2, open triangles; Syn-SerB-3, closed squares; and Syn-SerB-4, open squares. (B) ΔgltA: LacZ, open circles; GltA, closed circles; and Syn-GltA-1, closed triangles. (C) ΔilvA: LacZ, open circles; IlvA, closed circles; Syn-IlvA-1, closed triangles; and Syn-IlvA-2, open triangles. (D) Δfes: LacZ, open circles; Fes, closed circles; Syn-Fes-1, closed triangles; Syn-Fes-2, open triangles; Syn-Fes-3, closed squares; Syn-Fes-4, open squares; Syn-Fes-5, closed diamonds; Syn-Fes-6, open diamonds; Syn-Fes-7, ×; and Syn-Fes-8, +.

Michael A. Fisher, et al. PLoS One. 2011;6(1):e15364.
4.
Figure 2

Figure 2. Rescue of E. coli auxotrophs by de novo proteins.. From: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth.

(A) E. coli strain BW25113 Δfes was transformed with a control plasmid overexpressing LacZ (left half), or with a library of plasmids encoding the collection of novel proteins (right half). Cells were plated on M9-glucose supplemented with 50 µM IPTG, 30 µg ml−1 kanamycin, and 34 µg ml-1 chloramphenicol. Cells transformed with the control plasmid fail to grow, whereas cells transformed with the library yield occasional colonies. (B) Similar results for ΔilvA. (Similar results were also observed for ΔserB and ΔgltA – data not shown.) (C) Re-transformation of ΔilvA cells with a single sequence, syn-ilvA-1. A similar number of colonies grow on M9-glucose (left) as on LB (right). Results for other sequences were similar to those shown in (C), and are summarized in Figure S1.

Michael A. Fisher, et al. PLoS One. 2011;6(1):e15364.
5.
Figure 1

Figure 1. Design of a collection of novel proteins and rescue of E. coli auxotrophs.. From: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth.

Binary pattern of polar (red) and nonpolar (yellow) residues designed to fold into 4-helix bundles (7, 8). Library construction is facilitated by the organization of the genetic code: Six polar residues (Lys, His, Glu, Gln, Asp, and Asn) are encoded by the degenerate codon VAN, and five nonpolar residues (Met, Leu, Ile, Val, and Phe) are encoded by the degenerate codon NTN. Combinatorial turn positions are encoded by the degenerate codon VRS, which encodes Gln, Glu, Asn, Asp, His, Lys, Arg, Ser, and Gly. (V = A, G, or C; R = A or G; N = A, G, C, or T). Circles with letters indicate fixed residues, and empty circles indicate combinatorially diverse positions. The theoretical diversity of this library is 522×634×812 = 5×1052. The substantially smaller experimental library (1.5×106 synthetic genes cloned on a high expression plasmid) was transformed into a strain of E. coli in which an endogenous gene had been deleted (green X). Colony formation on minimal media indicates a novel sequence (purple) enables cell growth under selective conditions.

Michael A. Fisher, et al. PLoS One. 2011;6(1):e15364.

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