Gibson assembly reaction master mix

Example 3

The protein sequence for the Populus alba was obtained from GenBank and the full gene, codon optimized for E. coli was purchased from Eurofins MWG. This DNA was used as a template for amplification of the gene using primers 1 and 2 (see Table 1) and Phusion polymerase (NEB) with an annealing temperature of 45° C. The vector backbone of pBBR1MCS3-pBAD was generated with primer 3 and 4 (see Table 1) and with Merck Millipore KOD polymerase with annealing temperatures of 50-55° C. The two fragments were ligated using NEB Gibson Assembly reaction master mix as per the manufacturer's recommended protocol. The ligation mix was transformed into chemically competent E. coli NEB5α and correct clones verified via a combination of colony PCR and sequencing with primers 5 and 6 (see Table 1). Subsequently the whole construct was sequenced by MWG-Eurofins using primers 7-16 (see Table 1). A single verified construct was taken forward for further work and designated pBBR1-ISPS.

Example 2

The protein sequence for the Populus alba was obtained from GenBank (BAD98243.1) and the full gene (with an additional promoter and terminator), codon optimized for E. coli was purchased from Eurofins MWG (SEQ ID NO:52). This DNA was used as a template for amplification of the gene using primers 1 and 2 (see Table 1) and Phusion polymerase (NEB) with an annealing temperature of 45° C. (the open reading frame (ORF) generated lacked the native plasmid tag; this ORF corresponds to nucleotides 168-1865 of SEQ ID NO:52). The vector backbone of pBBR1MCS3-pBAD was generated with primer 3 and 4 (see Table 1) and with Merck Millipore KOD polymerase with annealing temperatures of 50-55° C. The two fragments were ligated using NEB Gibson Assembly reaction master mix as per the manufacturer's recommended protocol. The ligation mix was transformed into chemically competent E. coli NEB5α and correct clones verified via a combination of colony PCR and sequencing with primers 5 and 6 (see Table 1). Subsequently the whole construct was sequenced by MWG-Eurofins using primers 7-16 (see Table 1). A single verified construct was taken forward for further work and designated pBBR1-ISPS (see FIG. 2A; SEQ ID NO:15)