BioMed Research International / 2014 / Article / Tab 1

Research Article

C-Terminal Domain Swapping of SSB Changes the Size of the ssDNA Binding Site

Table 1

Primers used for construction of plasmids.

OligonucleotidePrimer

1 KpSSB-NdeI-NGGGCATATGGCCAGCAGAGGCGTAAAC
2 KpSSB-XhoI-CGGGCTCGAGTTAGAACGGGATGTCGTC
3 StSSB-NdeI-NCTGAACATATGGCCAGCAGAGGCGTAA
4 StSSB-XhoI-CTGGAACTCGAGTTAGAACGGAATGTCG
5 PaSSB-NdeI-NTTGCTCATATGGCCCGTGGGGTTAACA
6 PaSSB-XhoI-CTTGCACTCGAGTTAGAACGGAATGTCG
7 KpSSB(D91E/Q92L-SacI)-NAAGTGGACCGAGCTCTCCGGTCAGGACA
8 KpSSB(D91E/Q92L-SacI)-CGTCCTGACCGGAGAGCTCGGTCCACTT
9 StSSB(D91E/Q92L-SacI)-NAAGTGGACCGAGCTCAGTGGCCAGGAA
10 StSSB(D91E/Q92L-SacI)-CTTCCTGGCCACTGAGCTCGGTCCACTT
11 PaSSB(G90E/Q91L-SacI)-NAAGTGGCAGGAGCTCGACGGTCAGGAT
12 PaSSB(G90E/Q91L-SacI)-CATCCTGACCGTCGAGCTCCTGCCACTT
13 KpSSBnStSSBc(E91D/L92Q)-NAAGTGGACCGATCAGAGTGGCCAGGAA
14 KpSSBnStSSBc(E91D/L92Q)-CTTCCTGGCCACTCTGATCGGTCCACTT
15 KpSSBnPaSSBc(E91D/L92Q)-NAAGTGGACCGATCAGGACGGTCAGGAT
16 KpSSBnPaSSBc(E91D/L92Q)-CATCCTGACCGTCCTGATCGGTCCACTT

A fragment containing the coding sequence of KpSSB, StSSB, and PaSSB (with the stop codon) was cloned into the pET21b vector (using Primers 1–6). During the process, NdeI and XhoI restriction sites were introduced at the 5′-end and the 3′-end of these genes, after which they were ligated into the pET21b vector. To obtain the vectors for expression of the chimeric proteins KpSSBnStSSBc and KpSSBnPaSSBc, pET21b-KpSSB (Primers 7 and 8), pET21b-StSSB (Primers 9 and 10), and pET21b-PaSSB (Primers 11 and 12) vectors were mutated to create a desired SacI site. The D91E/Q92L-engineered pET21b-KpSSB vector, the D91E/Q92L-engineered pET21b-StSSB vector, and the G90E/Q91L-engineered pET21b-PaSSB vector were cut at NdeI and SacI sites. Subsequently, the KpSSBn, StSSBc-pET21b, and PaSSBc-pET21b fragments were purified. KpSSBn was ligated with StSSBc-pET21b and PaSSBc-pET21b fragments to generate the engineered pET21b-KpSSBnStSSBc and pET21b-KpSSBnPaSSBc vectors. To avoid artificial residues, positions 91 and 92 of the two plasmids were mutated back (Primers 13 to 16) to obtain pET21b-KpSSBnStSSBc and pET21b-KpSSBnPaSSBc vectors. Thus, pET21b-KpSSBnStSSBc and pET21b-KpSSBnPaSSBc will express KpSSB1-91 fused StSSB92-176 and PaSSB91-165, respectively. These plasmids were verified by DNA sequencing. Underlined nucleotides indicate the designated site for mutation or the restriction site.