Figure 4

Targeted integration of BglBrick parts into the E. coli genome. (A) A variety of basic parts were used to create two methyltransferase expression devices targeting BamHI and BglII restriction sites (parts J72007 and J72013, respectively). Each device was recombined into the genome of strain MC1061 by Φ80 att site integration, resulting in BamHI- and BglII-methylating strains (parts J72015 and J72014, respectively). (B) Sample experimental design for genomic integration of CRIM plasmids. Circuit components are color-coded and graphically represented relative to (A) and the top of (B) for easy identification. (1) Host strain MC1061 with unmodified genomic Φ80 att sites is transformed with temperature sensitive helper plasmid pInt80-649 (part J2008) and selected on ampicillin plates. (2) Cells are re-transformed with CRIM plasmid (part J72007), which replicates as a high-copy R6K plasmid employing the pir gene provided by the helper plasmid. (3) The CRIM plasmid inserts into the genome by recombination with the Φ80 att site employing the int gene from the helper plasmid. (4) Helper plasmid is cured by growth at 43°C. (5) Helper plasmid pCP20 encoding Flp recombinase is introduced by transformation and the R6K origin and chloramphenicol genes are excised from the genome by recombination of FRT sites. (6) Helper plasmid is cured by growth at 43°C, resulting in the final product containing a genomically-integrated BglBrick part and a single FRT site. (C) Restriction mapping of plasmid J61148-J72011 isolated from BglII- and BamHI- methylating strains (parts J72015 and J72014, respectively) or DH10B (control) confirms the appropriate protection.