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Figure 3 | Journal of Biological Engineering

Figure 3

From: Evolutionary principles and synthetic biology: avoiding a molecular tragedy of the commons with an engineered phage

Figure 3

Phage design. The part of the T7 genome subjected to cloning is shown, drawn to scale (approximately 4.5kb shown of a 40kb genome). (T7415) The cloning vector, indicating the locations of the promoter (pink) in front of gene 10A, all ribosome binding sequences (RBS, blue dots), and the 3 phage genes in the vicinity of the cloning region. The T7 terminator TÏ• is shown in gray, and the two restriction sites used for cloning are indicated with black, vertical lines (R1 is for Eco RI, H3 is for Hind III). (T7E1) The endosialidase was cloned between the Eco RI and Hind III sites with its own RBS and a stop codon terminating translation of 10A. The T7 terminator was located downstream of the cloning. (T7E2) The endosialidase was cloned between the Eco RI and Hind III sites as an inframe fusion with 10A. but the only clones obtained contained an evolved stop near the end of 10A. The T7 terminator was shifted downstream of the insert. Stops are present at ends of all genes in all 3 genomes (10A, Endo, 11, and 12). The control phage T70 is the same as T7415 with a 15 amino acid S-tag extension between the Eco RI and Hind III sites (flanked by single amino acids).

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