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Genomic integration of flip-flop devices using the miniTn7 Biobrick Tool kit

An anticipated benefit of single-copy integration of Biobricks in the bacterial chromosome is the possibility of placing sophisticated regulatory devices in a context that closely resembles the natural situation (i.e., regulatory elements are generally designed to function optimally in single-copy). We predict that having regulatory devices in single-copy will improve their performance by balancing the concentration of cis-and trans-acting elements and diminishing the effect of noise-generating phenomena, such as uneven plasmid partitioning between daughter cells, that may cause these systems to drift from their expected behavior. To test this, we have successfully cloned the "basic" and "improved" flip-flop devices in the miniTn7BB-Gm minitransposon. The presence of the inserts was evidenced by fluorescence of colonies and cell pellets (Figure 8), and the constructs were verified by restriction analysis (data not shown). These constructs were inserted in the chromosome of E. coli by coelectroporation with the helper plasmid pTNS2 for functional testing. Correct integration at the attTn7 site was tested by colony PCR as described above (data not shown).

Figure 8. Fluorescent pellets containing the improved flip-flop device. Pellets of E. coli strains containing the improved flip-flop in the miniTn7BB-Gm transposon in high copy number (pUC18-based vector, left), or medium copy number (R6K-based vector, center), or not containing the flip-flop device (right).

Figure 9. Fluorescent pellets containing the basic flip-flop device. Pellets of E. coli strains containing the basic flip-flop in the miniTn7BB-Gm transposon in medium copy number (R6K-based vector, left), high copy number (pUC18-based vector, center), or not containing the flip-flop device (right). It is quite interesting that the basal state of the basic flip-flop is red in the medium copy number plasmid and green in the high copy number plasmid.