The cDNA CLONING Team was headed by Brian Fox, PhD, and was responsible for obtaining and manipulating cDNAs in advance of protein production, including the production of vectors designed for this purpose.
The success of structural genomics initiatives is critically dependent on the incorporation of targeted open reading frames (ORFs) into vectors suitable for protein production. Among the many structural genomics groups using Gateway® recombination, CESG previously developed and used the Gateway® method to clone ~3500 eukaryotic ORFs. As part of this effort, a customized modular vector backbone was created to allow efficient swap of antibiotic resistance markers, protein tags, linker regions, and protease sites. Some of the best variants of these production vectors, available by material transfer agreement with the University of Wisconsin, are described.
To facilitate expansion of this vector set to include wheat germ cell-free and other expression platforms, we evaluated Flexi®Vector, a restriction enzyme/ligation based cloning system recently developed by Promega Corporation (Madison, WI). This system offered the advantages of high-throughput cloning of PCR products directly into an expression vector and serial transfer of the sequenced verified ORFs from the first vector to others. Here we report a comparison of Gateway® recombination cloning system and the Flexi®Vector restriction-based cloning system.
Cloning protocols for each system were conducted in parallel for 96 different target genes from PCR through the production of sequence verified expression clones. The shorter nucleotide sequences required to prepare the target ORFs for Flexi®Vector cloning allowed a single-step PCR protocol, resulting in fewer mutations relative to the Gateway® protocol. Furthermore, through initial cloning of the target ORFs directly into an expression vector, the Flexi®Vector system gave time and cost savings compared to the CESG protocol originally developed for the Gateway® system. Within the Flexi®Vector system, genes were transferred between four different expression vectors. The efficiency of gene transfer between Flexi®Vectors depended on including a region of sequence identity adjacent to one of the restriction sites. With the proper construction in the flanking sequence of the vector, gene transfer efficiencies of 95-98% were obtained. Detailed protocols developed for the Flexi®Vector method, the current catalog of vectors developed for this project, and opportunities for multiplexed cloning and expression studies are presented.
There are strengths and weaknesses inherent to any cloning system. The cloning steps in the Gateway® system are highly efficient, and there are a wide variety of vectors available due to the length of time this system has been in use. However, the requirement for an initial, non-productive cloning step and the long primer sequences required to encode the recombination sites are drawbacks to this system. In the Flexi®Vector system, the initial cloning step inserts the target gene into an expression vector and the short flanking nucleotide sequences can be added in a single PCR step. As demonstrated here, the efficiency of Flexi®Vector cloning can match or exceed that of recombination cloning for both initial capture of PCR products and for transfer between different vector combinations. These advantages lead to savings in time and cost, and fewer mutations present in the expression clones. There currently fewer vector options available for the Flexi®Vector system than for Gateway® and target genes must be screened for the presence of the Sgf I and Pme I sites.
We conducted a high-throughput Plasmid DNA screen to determine the presence of insert in both our Entry and Expression plasmids. A robot-aided protocol was developed whereby the colonies are picked into 96-well growth blocks containing CircleGrow™ media and the appropriate antibiotic. This is grown overnight at 37°C with vigorous shaking. The next morning plasmid DNA is isolated with the use of QiaRobot 8000. This DNA is then used as a template in PCR using universal vector primers that flank the insertion site. The PCR products are analyzed on E-gels 96 (Invitrogen) and positive clones moved down the pipeline.