Interaction of TrwC_R with Rep-like substrates.

<p>(A) SDS-PAGE of oligonucleotides with R388wt or Rep-Like structures, when incubated with TrwC_R. 6 μM TrwC_R was incubated with 15 μM of different oligonucleotides. The reaction products were separated by electrophoresis in 12% SDS-PAGE gels. Lane 1, no oligonucleotide; Lanes 2 and 3, R388wt oligonucleotides W(25+18) and W(14+14), respectively; in subsequent lanes, TrwC_R was incubated with Rep-like oligonucleotides. The S length of Rep-like substrates (in green), varies from two to eleven nucleotides. Lane 4, H(14+8), Lane 5, H(14+10), Lane 6, H(14+12); Lane 7, H(14+13), Lane 8, H(14+14); Lane 9, H(14+15); Lane 10, H(14+17). In the center chart, percentage of bound complexes were calculated in three separate experiments such as that shown in (A). (B) Increasing amounts of TrwC_R were incubated with wt oligonucleotide W(25+8) (red shift, lanes 1 to 8) or Rep-like hairpin H(14+14) (green shift, lanes 9–16). Lanes 1 and 9, no protein added; 2 and 10, 42 nM of TrwC_R; 3 and 11, 85 nM; 4 and 12, 210 nM; 5 and 13, 420 nM; 6 and 14,850 nM; 7 and 15, 4,2 μM, 8 and 16, 8,5 μM.</p

Strand transfer reaction catalyzed by TrwC_R of oligonucleotides with the Rep-like and Reverse-like layout to the W(25+0) oligonucleotide.

<p>Strand transfer reaction catalyzed by TrwC_R of oligonucleotides with the Rep-like and Reverse-like layout to the W(25+0) oligonucleotide.</p

Mobilization frequencies of synthetic <i>ori</i>T-plasmids.

<p>Mobilization frequencies obtained using pSU2007 as helper plasmid are expressed as number of transconjugants per donor cell. Values are the average of three experiments. The relative position of the secondary structure elements of the assayed synthetic <i>oriT</i>s is shown. R388 wt <i>oriT</i> has four IR that are binding sites for the relaxase and nicking-accessory proteins TrwA and IHF. TrwC binds IR₂ and the <i>nic</i> site (<i>sbc</i>, red), TrwA binds IR₄ (<i>sbaB</i>, green) while IHF binds IR₃ (<i>ibs</i>, blue). The distance between secondary structure elements is not shown at scale.</p

Scheme of relaxase and replicase DNA targets.

<p>(A) The relaxase DNA substrate contains an IR, defined by a Distal arm (D) and a Proximal Arm (P), that shape a hairpin structure. The <i>nic</i> site (N) is located between a U-turn (U) sequence and a ssDNA strand (S) that is tethered to the relaxase after cleavage. (B) The DNA substrate cleaved by replicases has the Distal arm (D) located downstream from <i>nic</i> (N), which allows stem-loop formation. Rep-like substrates for relaxases were designed by displacing the D sequence to the 3’ end of S of their original wt substrate. (C) Scheme depicting the cleavage reaction of the wt substrate. Upon binding, relaxase bends its target in order to localize the <i>nic</i> site (N) within its active center. In presence of a divalent cation, the relaxase cleaves <i>nic</i> (blue arrowhead) and remains covalently bound to the 5’-phosphate of S, downstream from N. (D) Scheme depicting the cleavage reaction of the Rep-like substrate. The stem is bound by the relaxase, and the loop is located within the DNA binding cleft. Thus, the relaxase cleaves the scissile nucleotide within a stable cruciform (blue arrowhead). After cleavage the relaxase will be tethered to S and D. P is shown in yellow and U in purple. Blue triangles show the position of the <i>nic</i> site.</p

Cleavage activity of TrwC_R on different oligonucleotides.

<p>Cleavage activity of TrwC_R on different oligonucleotides.</p

Novel designs for DNA substrates of model single-Y relaxases.

<p>(A) SDS-PAGE of MobA_R_RSF1010 with its targets. 7 μM MobA_R was incubated with 15 μM of different oligonucleotides. Lane 1, MobA_R. Lanes 2 and 3, wt oligonucleotide WQ(30+7) and a substrate that lacks the upper-hairpin nucleotides of the IR WQ(23+7). Lanes 4, 5 and 6, Rep-like oligonucleotides HQ(16+16),HQ(16+19) and HQ(16+22), respectively. Lanes 7, 8 and 9 reverse substrates RQ(8+28), RQ(8+34) and RQ(8+40). Lane 10, molecular weight ladder. (B) SDS-PAGE of TraI_R_RP4 with its targets. 1.5 μM TraI_R was incubated with 15 μM of different oligonucleotides. Lane 1, TraI_R; lane 2, wt substrate WP(15+6); lane 3, wt substrate WP(24+8); lane 4, reverse substrate RP(8+24) and lane 5, Rep-like substrate HP(14+14). Molecular weight ladder is shown on Lane 6. Bar graphs with the quantification of covalent complexes are shown below the SDS-PAGE gels. Data showed mean±s.d. of three independent experiments.</p

Interaction of TrwC_R with Reverse substrates.

<p>(A) Reverse substrates were designed by swapping the 5’ region of the <i>nic</i> site to the 3’ end. This designed DNA substrate possesses the complete inverted repeat (D-P) at the 3’ end of the <i>nic</i> site (N). Either the U or the S lengths were tuned to allow the correct location of the hairpin within the relaxase binding domain. (B) Scheme depicting the cleavage reaction of the reverse substrate. Relaxase binding to the reverse substrate allows both the hairpin and the single strand U-turn localize at the DNA binding cleft. This way the cleavage reaction forms a covalent complex of the relaxase with the region downstream of the <i>nic</i> site (blue arrowhead). Now the 5´side of <i>nic</i> do not contain the IR avoiding the re-ligation reaction. Color code is the same than <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152666#pone.0152666.g001" target="_blank">Fig 1</a>. (C) 12% SDS-PAGE of reverse oligonucleotides, when incubated with TrwC_R. 6 μM TrwC_R was incubated with 15 μM of different reverse oligonucleotides. Lane 1, no oligonucleotide; Lanes 2, R388wt oligonucleotide W(25+18). Lanes 3 and 4, Reverse substrates R(8+27) and R(8+24), both with U = 8 nt and S = 11nt or S = 8 nt respectively. Lane 5, R(7+27) U = 7; Lane 6, R(4+27) U = 4; Lane 7, R(1+27) U = 1 and Lane 8, R(0+27) U = 0. Lane 9, R(8+14), U = 8 P = 0. Lane 10, Molecular weight marker. Graph quantifying the percentages of covalent complexes is shown below the SDS-PAGE gel. Data show mean±s.d. of three independent experiments. Two asterisks indicate P-value<0.05 by two-sided student’s t-text.</p

Time evolution of NOR_1 according to a specific input profile.

<p>Logic case 1-1 is induced during the intervals [0…200] min and [400…500] min (A and B = 500 molecules -constant entry- during the interval). The case 0-0 domains during the rest of the 600 min. According to that profile we observe the deterministic oscillation of FimE/FimB (top graph) as well as the oscillation between the two possible plasmids in NOR_1 ( and ). The latter relation is shown deterministically (middle graph) and stochastically (bottom graph) (copy number = 5). Delays in response are due to input degradation times.</p

DPMT validation for MOB_C relaxases.

<p>A) Phylogenetic tree of MOB_C relaxase family. B) Alignment of the relaxase motifs used to design the MOB_C degenerate primers (C11-f+C11-r, continuous black; C12-f+C12-r, continuous dark grey). C) Amplicons obtained with primers for subfamily MOB_C11 (C11-f and C11-r). D) Amplicons obtained with primers for subfamily MOB_C12 (C12-f and C12-r). Symbols, colour codes and lanes as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040438#pone-0040438-g001" target="_blank">Figure 1</a>.</p

Deterministic and stochastic time evolution of the 1-1 logic case.

<p>These results show the simulation of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065986#pone-0065986-g001" target="_blank">Figure 1B</a> with input molecules ( and ) set up to 500 (each) ( =  = 500 molecules min⁻¹) and the copy number of the plasmid to 5. By monitoring (deterministically, upper graph, one run; stochastically, middle graph, ten runs) proteins Cre (in the Sender) and GFP (in the Receiver) we see how GFP production is initially triggered by those plasmids that have not been modified yet (). When Cre has been functioning long enough (t57 min in this simulation) the remaining GFP is only controlled by degradation rates, as no more fluorescent proteins are being expressed. Lower graph shows the stochastic evolution of over time, which determines the delay in displaying the correct output according to the NOR logic function (0 output for the 1-1 case).</p