Contributed by John M. Sedivy, Department of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar Street, New Haven, CT 06510.
Genes of higher eukaryotes can be hundreds of kilobases in size. The need to rapidly clone and manipulate DNA molecules in that range has become acute in conjunction with the Human Genome Project. Cosmid vectors are constrained to 4045 kb by the packaging limit of phage lambda. Recently, three systems have been developed that extend this range: yeast artificial chromosome (YAC)-based vectors (>500 kb limit),1 phage P1-based vectors (100 kb limit),2 and E. coli F factor-based vectors (limit as yet untested).35 YAC clones have proven very successful, but are difficult to manipulate due to the extreme fragility of large linear DNA molecules. Bacterial episomes, being supercoiled, dont suffer that constraint. Unfortunately, F factor vectors have been limited by the low efficiency of transformation of large DNA molecules into E. coli. Recently, through the use of electroporation, efficiencies of 1010 transformants/g DNA of small plasmids have been achieved. A systematic analysis of large DNA molecules has been reported,5 and is summarized here.
Materials and Methods
E. coli strain JS46 was grown at 37 C in LB broth; in mid-log phase (OD600=0.6) flasks were placed in ice water and chilled for 15 minutes. Cells were harvested, washed in ice-cold 1 mM HEPES, pH 7.0 (first wash 1x, second wash 0.5x of original culture volume), and resuspended at 1011 cells/ml in 10% glycerol. 40 l aliquots were flash-frozen in dry ice/ethanol and stored at -70 C. Cells were thawed on ice, DNA (2 l in TE) was added, and transferred to ice-cold 0.2 cm gap Bio-Rad electroporation cuvettes. A single pulse was delivered (Gene Pulser II apparatus used with the Pulse Controller II) at 25 F, 2.50 kV and 200 ohms (time constant=4.54.7 msec). Cells were immediately resuspended in 1 ml of SOC (room temperature) and incubated at 37 C for 45 minutes. Dilutions were plated on LB plates containing appropriate antibiotics. Plasmid DNA concentrations were determined by absorbance readings at OD260 and verified by gel electrophoresis.
We have investigated the relationship between plasmid size and electroporation efficiency in E. coli and found that even very large plasmids can be efficiently transfected. Six plasmids were used: pUC19 (2.6 kb),7 cosmid tsA/Y/1d4 (44 kb),8 and F factor clones pMBO52 (52 kb), pMBO74 (74 kb), pMBO99 (99 kb) and pMBO136 (136 kb).3 The average efficiencies (number of colonies per g DNA) were: pUC19, 2 x 109; tsA/Y/1d4, 6.1 x 107; pMBO52, 6.6 x 106; pMBO74, 3.2 x 106; pMBO99, 2.7 x 106; pMBO136, 1.7 x 106 (Figure 1). The data points can be fitted to a straight line when both axes are plotted on a logarithmic scale. The relation can be written as E=1.2 x 106M-0.4248, where E is efficiency (colonies/fmole), and M is plasmid size (kb).
Supercoiled and relaxed episomes were electroporated with equal efficiency. Rearrangements incurred during electroporation were analyzed at the level of restriction digestion; none were detected. To exploit these observations we have constructed a novel mammalian- E. coli shuttle vector, designated pFRS1 (F Replicon Shuttle, Figure 2), which contains three functional units: 1) the E. coli F factor origin of replication, partitioning, and copy number control region, 2) a marker selectable both in E. coli and mammalian cells (the neo cassette from plasmid pRSVneo), and 3) the polylinker from the pBluescript SK cloning vector. The vector stably confers resistance to kanamycin in E. coli, and resistance to G418 in mammalian cells. The plasmid is approximately 12 kb in size, and, like its F factor parent, is maintained at low copy number.
The new vector system should accept inserts well in excess of 100 kb. The efficiencies of electroporation are well above the minimum required to construct representative libraries of complex eukaryotic genomes. At this time, the only vector system available for the cloning of very large DNA fragments uses YAC vectors. E. coli has several advantages over yeast as an organism for the passive propagation of foreign DNA. E. coli grows faster, DNA can be rapidly and efficiently extracted, and yields, even of single copy plasmids, are significantly higher. In addition, circular supercoiled DNA molecules are physically quite stable and easy to handle. The most crucial issue, however, is the genetic stability of foreign DNA. Use of E. coli offers the distinct advantage of the very considerable body of knowledge concerning its recombination pathways. In several instances the instability of foreign DNA in E. coli has been overcome by the judicious choice of appropriate, multiply marked, recombination-deficient strains.911
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pBluescript is a registered trademark of Stratagene Cloning Systems.
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