ble 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 transformed into new competent cells. Cells were grown on LB agar containing 20 μg/mL ampicillin to select for transformants that contained a recombined BPP plasmid, and 40 μg/mL IPTG to induce pLac-driven expression of mRFP. mRFP expression was detected under ultraviolet light and photographed using a BioRad imager.
Availability and requirements
Vector BBa_J64100 was generously provided by J. Tabor. KAH, WLH, JOD and EZ were supported by HHMI grant 52005120. SR was supported by a grant from iGEM at the Massachusetts Institute of Technology. ADB, MLB, TLB, ELJ KJM, BJO, JLP, LHH, and TTE were supported by the Missouri Western State University Summer Research Institute. LJH and AMC worked pro bono. KAH, AMC, LJH, and TTE are members of the Genome Consortium for Active Teaching . We thank the reviewers for their help with improving the final manuscript.
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