Constructing pIL-SVnisBTC and Derivatives

The plasmid pIL-SVnisBTC was generated from pIL-SV and pIL3BTC^{31 (link)}. The plasmid pIL3BTC was digested with the restriction enzymes NotI (NEB) and BstXI (NEB) to receive a fragment BTC containing the genes nisB, nisT and nisC. Next, pIL-SV^{62 (link)} was also digested with NotI and BstXI (pIL-SV**). The fragment BTC and pIL-SV** were ligated with T4-ligase (NEB) and transformed into E. coli DH5α. The sequence of the construct pIL-SVnisBTC (Table S5) was verified by DNA sequencing (Microsynth Seqlab).
By using Phusion DNA polymerase (NEB) with the appropriate primer pairs (Table S4) the gene deletions of nisB, nisC or nisT were performed to generate pIL-SVnisBTC derivatives. Subsequently, the linearized vectors were ligated with T4-ligase (NEB) and transformed into E. coli DH5α. The sequence of the constructs (Table S5) were verified by DNA sequencing (Microsynth Seqlab).
To generate nisT_H551A and nisC_H331A mutants, a polymerase chain reaction using PfuUltra II Fusion DNA polymerase (Agilent Technologies), the template pIL-SVnisBTC and the appropriate pair of oligonucleotides (Table S4) was performed according to standard procedures. The sequence of the new constructs (Table S5) were verified by DNA sequencing (Microsynth Seqlab).