etA(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 (Table 1) and the HinLVA expression plasmid were combined in 5 μL sterile distilled H2O and co-transformed into Z-competent JM109 chemically competent cells according to the manufacturer's protocol (Zymo Research). Cells were grown at 37°C on LB agar selective media (20 μg/mL ampicillin, 50 μg/mL chlroamphenicol).
Detection of DNA inversions
Inversions of single hixC-flanked DNA segments were detected by restriction digests (Fig. 3). Plasmid DNA was purified from each culture (Promega Wizard and Zymo Zyppy miniprep systems), digested with NheI, and resolved by agarose (Fig. 3a) or polyacrylamide (Fig. 3b) gel electrophoresis. Inversions of adjacent hixC-flanked segments were detected by multiplex semiquantitative PCR (sqPCR) (Fig. 4). 11 hours following transformation, 46 visible co-transformant colonies were picked from LB agar selective media and subjected to whole cell multiplex sqPCR (95°C, 10 min.; 95°C, 30 sec., 60°C, 30 sec., and 72°C, 15 sec. for 26 cycles). Each colony was suspended in 60 μL 1× PCR mix (Promega Green master mix plus 0.7 μM primers pLacR 5'-GAATCGGCCAACGCGCGGGG, pLacF 5'-GTTTCCCGACTGGAAAGCGG, TetR 5'-GTAGAGGATCCACAGGACGG and TetF 5'-TCGTAGGACAGGTGCCGGCA). 0.1 pmol of an equimolar mix of all 8 two-pancake BPP plasmids or 0.1 pmol of the (-2, 1) BPP plasmid alone was used as the template in a control PCR reaction. During PCR, an 11 μL aliquot was collected from each reaction after cycles 18, 20, 22, 24 and 26. Samples were electrophoresed on a 1.5% agarose gel and photographed under ultraviolet light using a BioRad imager. Band intensities were quantified using BioRad Quantity One imaging software. The detectable band threshold was set at 10,000 (after background subtraction).
To detect simultaneous flipping of two DNA segments (described in Fig. 5) single co-transformed colonies were picked from agar plates and grown in selective liquid media (20 μg/mL ampicillin, 50 μg/mL chlroamphenicol). To eliminate the Hin plasmid after flipping, plasmid DNA was purified from the liquid cultures and tran