A simple, flexible and efficient PCR-fusion/Gateway cloning procedure for gene fusion, site-directed mutagenesis, short sequence insertion and domain deletions and swaps

Abstract

Background: The progress and completion of various plant genome sequencing projects has paved the way for diverse functional genomic studies that involve cloning, modification and subsequent expression of target genes. This requires flexible and efficient procedures for generating binary vectors containing: gene fusions, variants from site-directed mutagenesis, addition of protein tags together with domain swaps and deletions. Furthermore, efficient cloning procedures, ideally high throughput, are essential for pyramiding of multiple gene constructs.

The Enzyme-Like Domain of Arabidopsis Nuclear β-Amylases Is Critical for DNA Sequence Recognition and Transcriptional Activation

“…Site-directed mutagenesis and domain swapping were conducted by a Gateway technology (Invitrogen) based method described before (Atanassov et al, 2009). Primers used are listed in Supplemental Table 6.…”

APETALA2/ETHYLENE RESPONSE FACTOR and basic helix–loop–helix tobacco transcription factors cooperatively mediate jasmonate‐elicited nicotine biosynthesis

“…G-box and GCC motif synthetic promoter sequences were PCR amplified with Gateway attB sequences, after which they were inserted in front of the luciferase gene into the Gateway destination vector pGWL7. The construct comprising both the G-box and GCC motif tetramers in front of the luciferase gene was generated with a PCR-fusion/Gateway cloning procedure (Atanassov et al, 2009) using the above plasmids as the starting template for PCR.…”

Seamless GFP and GFP-Amylase Cloning in Gateway Shuttle Vector, Expression of the Recombinant Proteins inE. ColiandBacillus Megateriumand Assessment of the GFP-Amylase Thermostability

“…PCR fusion/ Gateway cloning and expression clones the PcR fusion/Gateway cloning followed the procedure described by Atanassov et al (1). For cloning of the pXG expression clone, the two fused fragments, XylR/pXylA' and GFP, were first PCR amplified from plasmid pXZ, using the primers M13F (5'-gtaaaacgacggccagt, universal primer) and XGR (5'-agttcttctcctttgctcattgtacatttccccctttga, fusion primer), and from plasmid pGFPZ, using the XGF (5'-tcaaagggggaaatgtacaatgagcaaaggagaagaact, fusion primer) and M13R (5'-caggaaacagctatgac, universal primer).…”

“…tag) sequence insertions, gene domain deletion or swap of domains between genes. the protocols for these applications are described in details by Atanassov et al (1) and could be directly applied for construction of new expression vectors based on the pMMc shuttle vector. the range of the cloning scenarios described in this study could be largely expanded utilizing three features of the Gateway cloning system and PcR fusion cloning procedure as follows:…”

“…megaterium expression vectors offering application of sequence-independent recombinational cloning (16). the combination of the latter with a seamless DnA fusion method could further empower the capacity for recombinant gene cloning and expression with such vectors (1).…”