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Applications of BBa_J31001

Inversion of HixC-flanked DNA in the presence of HinLVA HinLVA has been assembled with a pLac promoter and RBS (see BBa_S03536) to create a HinLVA expression casette. We observe inversion of HixC-flanked segments of DNA in the presence of this casette. Figure 2 shows the sizes of predicted NheI restriction fragments for different conformations of two HixC-flanked DNA segments. A construct carrying the pBad promoter in the forward orientation (BBa_S03564) yields a 200 bp fragment (Figure 3, lane 2), while the reverse orientation (BBa_S03565) yields a larger 300 bp fragment (Figure 3, lane 3). In the presence of the HinLVA expression casette, the construct carrying pBad in the forward orientation shows restriction fragments for both orientations (Figure 3, lane 4). Similar results are seen for a construct carrying a HixC-flanked RBS-Tet segment (BBa_J3103) (Figure 4). The occurrence of the forward and reverse orientations in roughly equal proportions suggests that inversion has reached a steady state. Inversion occurs without IPTG induction of pLac-Hin. This may be caused by Hin expression via read-through from the vector backbone or a leaky pLac promoter.
Figure 2. Predicted NheI fragments for different conformations of the different HixC-flanked DNA fragments tested (not drawn to scale).	Figure 3. An NheI digest detects Hin-mediated flipping of a HixC-flanked pBad promoter.	Figure 4. An NheI digest detects Hin-mediated flipping of a HixC-flanked coding region (RBS-Tet).

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