FAQspeak

What is the largest insert a Promega plasmid vector will accept?

Plasmid vectors do not, in general, have any theoretical limit to the size of insert that they will accept. In nature, many very large plasmids exist in various bacterial species. In laboratory experiments, large constructs are routinely created in cosmid and PAC vectors and are stably maintained in E. coli. These constructs have the same basic structural elements, (e.g., origins of replications, antibiotic resistance markers) as standard cloning plasmids, such as the pGEM^®-T Vectors (Cat.# A3600). Cosmids will typically accept inserts of ~30-40kb (1); PAC vectors, up to ~100kb (2). With the exception of the pALTER^® Vector series, all of Promega's cloning vectors have pMB1 origins of replication. This origin comes from pUC vectors and is a derivative of the ColE1 origin of pBR322, which is the same origin as is used in standard Cosmid and PAC vectors. In fact, Tao and Zhang (3) have reported stable transformation of pGEM^®-11Zf(+/-) with inserts of up to 100kb. Although it appears that Promega vectors will, in general, accept inserts of this magnitude, there are obviously some difficulties associated with cloning large inserts, which stem from three different aspects of cloning: transformation, stable propagation and ligation. Each of these areas is discussed in more detail below.

Transformation: Transformation efficiency tends to go down as the size of the DNA being transformed goes up. This means that larger constructs are more difficult to introduce into bacterial host cells. It has been shown that transformation efficiency (transformants per microgram of DNA) of electrocompetent cells is inversely proportional to plasmid size in the range of 3-14kb (4), with 2.9kb plasmids yielding approximately 60 times more colonies per microgram than 13.6kb plasmids. Even taking into account the fact that the smaller plasmid will have more molecules per microgram, there is a difference of about 10 times in their molar transformation efficiencies. A mutation in the deoR gene of E. coli has been found to be helpful in increasing transformation efficiency of large plasmids. A mutation in this gene allows constitutive synthesis for genes involved in deoxyribose synthesis. The strain DH10B™, available from Invitrogen-Life Technologies, Inc. (www.invitrogen.com) is commonly used for transformation of large constructs (5).

In addition, Stratagene has developed a new phenotype, Hte, which apparently increases transformation efficiency of large constructs significantly (www.stratagene.com).

The lowered transformation efficiency of large plasmids will compound any other difficulties in cloning plasmids with large inserts. There are several methods to help alleviate this problem.

• Be sure to use high-efficiency competent cells.
• Consider using electroporation. Electroporation tends to have higher transformation efficiency than chemical transformation at all plasmid sizes.
• Decrease selection antibiotic concentration. Larger plasmids tend to have lower copy numbers than smaller plasmids, and thus antibiotic resistance genes on large plasmids are not as effective as those on smaller plasmids. In some cases decreased antibiotic concentration can improve growth of large plasmids.

Stable Propagation: Plasmid stability is dependent on a number of factors including expression of toxic gene products, positive selection pressure such as that due to antibiotic resistance genes, recombinogenic potential of sequences present in the plasmid, copy number of the plasmid, metabolic burden of plasmid maintenance, host strain genotype, and other factors.

The larger the plasmid, the greater the likelihood that it will contain sequences or other elements that will be selected against by the host bacterium. In addition, the larger the plasmid, the lower the copy number will be and the greater the metabolic burden it will place on its host.

Stability problems can be difficult to overcome. If a specific sequence is not stable, it can be helpful to grow the bacteria at a reduced temperature to reduce the rate of growth. If this is not successful, various different E. coli hosts may be tried. Generally, hosts are chosen at random according to what is available, but there are several strains (notably SURE^® cells from Stratagene and STBL2™ and STBL4™ cells from Life technologies, Inc.) that have been designed for increased stability of problematic plasmids and can be helpful for problematic clones.

For inserts that are not stable whatever is tried, we recommend that a low-copy-number plasmid be used. This will reduce the metabolic burden on the host and decrease expression of any potentially toxic proteins that may be produced.

Ligation: As the size of the vector and the insert increase it becomes increasingly unlikely for the ends to meet during ligation (6). In addition, the optimum total DNA concentration present during ligation changes as the sizes of the DNA changes (see Table 1).

Adapted from Reference 6.

References

1. Meyerowitz, E.M. et al. (1980) A new high-capacity cosmid vector and its use. Gene 11, 271-282.
2. Pierce, J.C., Sauer, B. and Sternberg, N. (1992) A positive selection vector for cloning high molecular weight DNA by the bacteriophage P1 system: Improved cloning efficacy. Proc. Natl. Acad. Sci. USA 89, 2056-2060.
3. Tao, Q. and Zhang, H.B. (1998) Cloning and stable maintenance of DNA fragments over 300kb in Escherichia coli with conventional plasmid-based vectors. Nucl. Acids Res. 26, 4901-4909.
4. Siguret, V. et al. (1994) Effect of plasmid size on transformation efficiency by electroporation of Escherichia coli DH5 alpha. BioTechniques 16, 422-426.
5. Sheng, Y., Mancino, V. and Birren, B. (1995) Transformation of Escherichia coli with large DNA molecules by electroporation. Nucl. Acids Res. 23, 1990-1996.
6. Dardel, F. (1988) Computer simulation of DNA ligation: Determination of initial DNA concentrations favouring the formation of recombinant molecules. Nucl. Acids Res. 16, 1767-1778.