Flexi vector cloning

Abstract

A protocol for ligation-dependent cloning using the Flexi Vector method in a 96-well format is described. The complete protocol includes PCR amplification of the desired gene to append Flexi Vector cloning sequences, restriction digestion of the PCR products, ligation of the digested PCR products into a similarly digested acceptor vector, transformation and growth of host cells, analysis of the transformed clones, and storage of a sequence-verified clone. The protocol also includes transfer of the sequence-verified clones into another Flexi Vector plasmid backbone. Smaller numbers of cloning reactions can be undertaken by appropriate scaling of the indicated reaction volumes.

Fig. 4.1

Escherichia coli expression vector pvp56k. (a) Linear map showing key features of the vector. (b) Sequence in the region near to the sgfi site. The nucleotide and encoded protein sequence of a portion of the linker between His8-MBP and the target is shown. The TEV protease site is ENLYFQA, where proteolysis occurs between the Q and A residues. After expression of the fusion protein, an N-terminal AIA-target is released by treatment with TEV protease. The identity of the next residues in the target is determined by the PCR primer design. (c) Sequence in the region near to the pmeI site, including the stop codon of the target gene.

Fig. 4.2

An example of 5′ coding and 3′ reverse complementary strand primers created for Flexi Vector cloning. (a) The 5′ primer consists of an exact match of the desired gene-specific sequence and an additional sequence encoding an sgfi site (34 nucleotides). (b) The 3′ complement reverse primer consists of an exact match of the gene-specific sequence including the stop codon, a primer-encoded stop codon and an additional sequence adding a pmeI site (33 nucleotides).

Fig. 4.3

Escherichia coli expression vector pvp68k. (a) Linear map showing key elements in the vector. (b) Sequence in the 5′ region near to the sgfi site. The first round forward PCR primer includes a gene-specific sequence and a portion of the TEV protease site (29 nucleotides). The second round forward PCR primer is a universal sequence that completes the TEV protease site and appends a 5′ sgfi sequence (34 nucleotides). The first round 3′ reverse complement primer includes a gene-specific portion, an additional stop codon and the pmeI site and a redundant ecori site (39 nucleotides). The second round reverse complement PCR primer is a universal sequence that duplicates the pmeI and EcoRI sites (24 nucleotides). After expression of the His8-MBP-target fusion protein, an N-terminal S-target can be released by treatment with TEV protease.

Fig. 4.4

A primer design example for expression of a native protein using Flexi Vector cloning. The promoter region is located upstream of the sgfi site and the start codon encoded by the 5′ primer.