cer (RE, BBa_J3101) , hixC (BBa_J44000) , RBS rev (BBa_J44001) and pBAD rev (BBa_J44002) were assembled from 20 – 60 bp DNA oligomers that were designed using the "Oligo Cuts Optimization Program" . EcoRI (5') and PstI (3') single stranded extensions were manually added to the terminal oligomers. An equimolar mix of single-stranded oligomers in 1× annealing buffer [100 mM NaCl; 10 mM Tris-HCl, pH 7.4] was incubated at 100°C for 5 minutes, then slowly cooled to ambient temperature in a water bath to produce double stranded DNA with EcoRI (5') and PstI (3') single stranded overhangs. The annealed DNA was ligated into linearized (EcoRI and PstI digested) cloning vector pSB1A2 using T4 ligase (Promega). Hin (BBa_J31000), HinLVA (BBa_J31001), and tetA(C) (BBa_J31007), and were cloned by polymerase chain reaction (PCR). Standard BioBrick prefix cloning sites (EcoRI, NotI and XbaI) or suffix cloning sites (SpeI, NotI and PstI) were included in the forward or reverse PCR primer, respectively. PCR amplicons were digested with EcoRI and PstI, electrophoresed in an agarose gel, purified, and ligated into a linearized cloning vector. Hin [GenBank: see Availability and requirements section for URL] was cloned from Salmonella typhimurium Ames strain TA100 genomic DNA using forward primer 5'-GCATGAATTCGCGGCCGCTCTAGATGGCTACTATTGGGTATATTC and reverse primer 5'-ATGCCTGCAGGCGGCCGCAACTAGTTAATTCATTCGTTTTTTTATAC. HinLVA was generated by PCR amplification of Hin (BBa_J31000) using forward primer 5'-TCTGGAATTCGCGGCCGCATCTAGAGATG and reverse primer 5'-CTGCAGGCGGCCGCTACTAGTATTAAGCTACTAAAGCGTAGTTTTCGTCGTTTGCAGCATTCATTCGTTTTTTTATAC containing a ssrA LVA degradation tag (based on gfp(down, LVA) ).tetA(C) was cloned from vector pSB1AT3 using forward primer 5'-GCATTCTAGATGAAATCTAACAATGCGCTCATC and reverse primer 5'-ATGCACTAGTTAGGTCGAGGTGGCCCGGC; the amplicon was cloned using a XbaI/SpeI digest. Reversed parts were generated by PCR amplification of MIT Registry parts using a forward primer containing SpeI and a reverse primer containing XbaI: tetA(C) rev (BBa_J31006) – forward 5'-ATGCACTAGTATGAAATCTAACAATGCGCTCATC and reverse 5'-GCATTCTAGATTAGGTCGAGGTGGCCCGGC; pLac rev (BBa_J31013) – forward 5'-ATGCACTAGTACAATACGCAAACCGCCTCTC and reverse 5'-GCATTCTAGAGTGTGTGAAATTGTTATCCGC; mRFP rev (BBa_J31008) – forward 5'-ATGCACTAGTATGGCTTCCTCCGAAGACGT and reverse 5'-GCATTCTAGATTAAGCACCGGTGGAGTGAC. Correct sizes and sequences of the genetic elements described above were confirmed by XbaI/SpeI double digestion and DNA sequencing. The constructs shown in Figure 2, the eight two-pancake BPP plasmids (Table 1), and the HinLVA expression vector were assembled using the standard BioBrick assembly method . Proper assembly of construction intermediates was confirmed by EcoRI/PstI double digestion. Fully assembled two-pancake BPP constructs (1, 2) (BBa_S03684), (-2, -1) (BBa_S03681), (1, -2) (BBa_S03685), (-2, 1) (BBa_S03679), (-1, 2) (BBa_S03687), (-2, 1) (BBa_S03680), (2, 1) (BBa_S03677), and (-1, -2) (BBa_S03688) were confirmed by sequencing. BPP constructs were inserted into low copy pSC101 vector pSB4A3 (Fig. 2) and subsequently tested in insulated high copy pMB1 vector pSB1A7. pLac-RBS-HinLVA-TT (BBa_S03536) was inserted into low copy ColE1 vector BBa_J64100 (from Jeffrey J. Tabor) to create the HinLVA plasmid (Fig. 2). The cloning vectors are described in .
Hin-mediated DNA recombination
Growth of transformed colonies on LB agar and in shaking liquid cultures took place overnight at 37°C. For single DNA segment flipping assays (Fig. 3), constructs containing the HinLVA gene on the same vector as the invertible segment (BBa_J44006 – pLac-RBS-HinLVA-TT-hixC-pBAD-hixC-RBS-tetA(C)-TT-RE or pLac-RBS-HinLVA-TT-pBAD-hixC-RBS-tetA(C)-hixC-TT-RE in vector pSB1A2) were transformed into JM109 chemically competent cells. After growth on selective media (LB agar plus 100 μg/mL ampicillin), single colonies were grown in liquid selective media (LB plus 100 μg/mL ampicillin) overnight. For the two-pancake stack flipping assay, 10 ng each of a plasmid containing a two-pancake stack (Ta