Supplementary figures, table and model code from Mathematical model of a serine integrase-controlled toggle switch with a single input

Figure S1, Figure S2, Figure S3; Table S1 with parameter values; Matlab code of the mode

Design of ZFRs, Z-sites and test substrates.

<p>(<b>A</b>) Cartoon showing a Tn<i>3</i> resolvase dimer bound to <i>res</i> site I. The red ovals represent the N-terminal (catalytic) and C-terminal (DNA-binding) domains. The motifs recognized by the C-terminal domains are yellow, and the central 12 bp segment (pink) is contacted by the catalytic domains.(<b>B</b>) Cartoon showing a ZFR dimer bound to a Z-site. The zinc finger domains are blue, the motifs they recognize are pale blue, and the ZFR linker peptide is green. Other features are as in <b>A</b>.</p

Recombination between non-identical sites.

<p>(<b>A</b>) Substrates containing a Ztn3Z site and a ZcasZ site are recombined by ZFR310. The annotation is as in the previous Figures. The band marked with an asterisk is thought to be single-stranded expression plasmid DNA. (<b>B</b>) Substrates containing a ZcasZ site and Tn<i>3 res</i> site I are recombined when ZFR310 and NM resolvase are co-expressed. (<b>C</b>) A substrate containing an Acas6B site and Tn<i>3 res</i> site I is recombined most efficiently when NM resolvase, ZFR^A320 and ZFR^B320 are co-expressed.</p

Mutant ZFRs selected for recombination of Zcas2Z or Zcas5Z sites.

<p>The Table shows mutants isolated from libraries of mutagenized ZFR300. The left-hand column gives the mutations, and the next column indicates the number of independent isolates. The other columns show the phenotype of each mutant in the MacConkey agar colony colour assay, using substrates with two identical ZcasZ sites as indicated. W, ‘white’ (pale-coloured) colonies; R, red colonies; M, mixtures of pale and red colonies. The letters in bold show the ZcasZ substrate (Zcas2Z and/or Zcas5Z) that was used in the screen from which the mutant was isolated. The four point mutants shown below the thick line were isolated by cloning appropriate fragments from the originally isolated multiple mutant (above the line). n.d., not done.</p

Casein gene sequences, Z-sites, and recombination assay.

<p>(<b>A</b>) Tn<i>3 res</i> site I (top line) and the seventeen TATA-containing sequences from the β-casein gene intron 1 (Genbank accession number X14711), aligned with the central TATA of site I. Sequences are numbered from the start of exon 1. The motifs in site I bound by the resolvase C-terminal domains are highlighted in yellow. Bases identical to site I within the central 12 bp of the casein sequences are highlighted in pink. The column on the right gives the number of bases identical to the central 12 bp of site I (the number before the slash is for the alignment shown, and the number after the slash is for the sequence aligned in the opposite orientation). The six ‘cas’ sequences, whose central 22 bp sequences were used in the recombination sites analysed in this study, are indicated. The 9 bp sequences flanking the central 22 bp are also shown (highlighted in blue); these sequences would be bound by the ZFR zinc finger domains if the genomic casein gene sequences were to be targeted as Z-sites (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019537#pone-0019537-g001" target="_blank">Figure 1B</a>). (<b>B</b>) Sequences of site I and Z-sites. The Z-site motifs recognized by the Zif268 domains are highlighted pale blue. The central 22 bp sequences of the ZcasZ sites are from the casein gene sequences shown in part <b>A</b>. Identities of the sequences to the central 12 bp of site I are highlighted pink. Note that the ZcasZ sequences are aligned here to maximize their matches with site I, so some are in the opposite orientation from part A. Motifs recognized by mutant Zif268 domains ZifA and ZifB are highlighted in magenta and cyan respectively. See text for further details. (<b>C</b>) Z-site substrate plasmids and colony colour assay. Recombination between the two Z-sites (boxes) deletes the <i>galK</i> gene, causing colonies to be pale rather than red on MacConkey-galactose indicator plates.</p

Mutant ZFRs selected for recombination of Zcas6Z sites.

<p>The Table shows the ‘second round’ mutants isolated from screens of ZFR310 mutant libraries (ZFR310 is ZFR300 with the mutation I77L), using a test substrate containing two Zcas6Z sites. The left-hand column gives the mutations, and the next column indicates the number of independent isolates. The other columns show the phenotype of each mutant in the MacConkey agar colony colour assay, using substrates with two identical ZcasZ sites as indicated. W, ‘white’ (pale-coloured) colonies; R, red colonies; M, mixtures of pale and red colonies.</p

Recombination of asymmetric Z-sites.

<p><b>(A)</b> Altered-specificity zinc finger domains target ZFRs to their cognate Z-sites. Substrates containing two identical Z-sites, with either A, B, or Z flanking motifs (Atn3A, Btn3B, Ztn3Z; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019537#pone-0019537-g002" target="_blank">Figure 2B</a>), were recombined by ZFR300 variants with alternative zinc finger domains as indicated immediately above each lane. (<b>B</b>) Efficient recombination at asymmetric (Atn3B) Z-sites requires two ZFR300 variants, with ZifA and ZifB specificity. The letters above each lane indicate the binding domains borne by the expressed ZFRs, with specificity for either Zif268 (Z), ZifA (A), or ZifB (B) motifs. exp(α), pACYC184-based ZFR expression plasmid (pαZFR); exp(β), pBR322-based ZFR expression plasmid (pβZFR). (<b>C</b>) Efficient recombination of AcasB site substrates requires co-expression of ZFR^A320 and ZFR^B320.</p

Selection of ZFRs to recombine casein gene sequences.

<p>(<b>A</b>) Activity of the progenitor ZFR (ZFR300) on Z-sites with central sequences from Tn<i>3 res</i> site I or the casein gene intron 1. The substrate plasmids each contained two identical Z-sites as indicated above the gel lanes. The numbers below each lane represent the percentage of recombinant plasmids in the recovered DNA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019537#s2" target="_blank">Materials and Methods</a>). Images of the ethidium-stained gels are greyscale-inverted, and a uniform background subtraction has been applied. A representative sector of a plate from the corresponding MacConkey-galactose assay is shown above each lane. Annotation: exp, ZFR expression plasmid; nr, non-recombinant substrate plasmid; rec, recombinant (<i>galK</i>⁻) plasmid. (<b>B</b>) Activity of the first round mutant I77L (ZFR310) on the same substrates as in <b>A</b>. (<b>C</b>) Activity of the second round double mutant I77L E132A (ZFR320) on the same substrates as in <b>A</b>.</p

Supplementary figure S1, Table S1 and Matlab code of the model from A simplified mathematical model of directional DNA site-specific recombination by serine integrases

Fig. S1: Comparison of the kinetics of the P x B(-R) reaction in minimal and full models; Table S1: Parameter values of the model; Matlab code of file