Methods and reagents: Ghost plasmid of pBluescript Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column discusses a ghostly band that haunts users of Stratagene's pBluescript vector. For details on how to partake in the newsgroup, see the accompanying box. Researchers using Stratagene's pBluescript phagemid vectors in combination with Escherichia coli host strain XL1-Blue have reported a contaminant within their plasmid preparations. An extraneous band is seen migrating at a position corresponding to approximately 2.0-2.2 kb of linear double-stranded DNA when a sample of enzyme-digested plasmid DNA is run on an agarose gel and stained with ethidium bromide. Some netters said that they observed an extra band, but that it migrates at different positions on the gel depending on the size of the cloned insert and the amount of plasmid DNA that is loaded onto the gel. Surprisingly, the band is not present in all plasmid preparations from different clones, or even from different preparations made from the same bacterial culture. The `ghost' band usually disappears upon transformation of a fresh batch of competent cells, only to return after several subcultures have been made; thus it has acquired the nickname `the Freddy Krueger plasmid' (after the haunting character in the movie A Nightmare on Elm Street). Like other ghostly visitors, the band normally does not interfere with further DNA manipulations and is therefore mostly ignored. It could simply be covalently closed circular (ccc)DNA that is recalcitrant to enzyme digestion. However, the exact source of this band is unknown, and some experimenters would like to ascertain the purity of their samples. One person wrote that the band was visible when stained with acridine orange. When excised from the gel, it was found to be resistant to many restriction endonucleases that normally recognize sites within the multiple cloning region of pBluescript. Both of these properties are consistent with it being at least partially single-stranded nucleic acid. Interestingly, the band recovered from an agarose gel is able to transform E. coli to ampicillin resistance. When plasmid DNA is extracted from the transformants and digested to linear form, a different band can be seen corresponding to the size of pBluescript (2.96 kb), leading to the conclusion that the contaminant could be a modified version of pBluescript. It is also possible that these transformants arose from a small amount of contaminating pBluescript DNA within the sample. Since many netters purify plasmids by the alkaline lysis procedure in combination with Promega's Magic [TM] mini-prep kit or with Qiagen purification columns, one possiblity is that the band appears when these methods are used in combination. Modified forms of plasmid DNA could arise from nicking, degradation or denaturation before or after binding to the specialized resin of mini-prep purification kits. For example, supercoiled plasmid molecules can be irreversibly denatured when exposed to harsh alkaline treatment for an extended duration, causing the cccDNA to form incongruent complementary base pairs. This would create a compact molecule capable of migrating faster than the linear plasmid during electrophoresis. The secondary structure could also be responsible for the observed resistance to endonuclease digestion. It is also possible that the enzyme resistance could be due to impurities passing from the binding resin of the column into the DNA sample when centrifuged for longer than normal or at speeds exceeding those recommended by the manufacturer. This could account for the additional observation that cloning vectors derived from the pUC series (other than pBluescript) show resistance to many common restriction ezymes when isolated using these techniques. Others raised the concern that the ghost might not be a supercoiled form of pBluescript, but an entirely unrelated plasmid inherent to the host strain XL1-Blue [1]. Sheryl L. White at the University of Vermont (slw@salus.med.uvm.edu) tried to sequence the ghost band from a recombinant derivative of pBluescript using T3, T7, M13 -20, and M13 reverse primers. In all cases, the patterns observed indicated that the primers did not hybridize to the template DNA, whereas a positive control sample of supercoiled pBluescript could easily be sequenced under the same conditions. Therefore, either the plasmid was not pBluescript, or at least the multiple cloning site (MCS) region of the derivative was missing. Some netters are convinced that the extra band is a product of the f1 origin of plasmid replication cloned onto pBluescript and that, since the band is mostly seen when E. coli XL1-Blue is used as host, it is responsible for creating the ghost band. Presumably, the molecule produced would be single-stranded viral coding-strand phagemid DNA made from aberrant recognition of the nick site at the f1 origin present on the vector. It is not known how this could occur without helper-phage infection, except that perhaps a portion of the gene product II (an endonuclease; for further details, see Ref. 3) from a segment flanking the intergenic region is present, and a partial gpII protein is made at low levels. As an argument against this theory, however, the 456 bp fragment containing the intergenic region of phage f1 used for the construction of the phagemid does not contain the open reading frame or promoter region of gene II (Ref. 2). It is entirely possible that XL1-Blue is contributing a factor(s) for the spontaneous initiation of coding strand replication, and that the ghost band could be any one of a number of replication intermediates or recombination products. If this is true, then the host-encoded factor(s) is probably expressed under some growth conditions, but not others, since the band is not always observed. However, the involvement of other host factors, such as transposon Tn10 residing on the F plasmid within XL1-Blue, cannot be excluded. Regardless of the exact source of the `ghost' plasmid, many netters would like to eliminate the band from their samples. To avoid it, pBluescript users are now trying to circumvent the use of XL1-Blue host whenever possible. If they cannot, they isolate their constructed recombinant plasmid DNA immediately after characterization, and then transform a different host strain for further manipulations or long-term storage. Most netters have found HB101 or DH5alpha to be an acceptable and often preferred substitute for XL1-Blue. References: [1] Bullock, W. O., Fernandez, J. M., and Short, J. M. (1987) BioTechniques 5, 376-379 [2] Short, J. M., Fernandez, J. M., Sorge, J. A., and Huse, W. D. (1988) Nucleic Acids Res. 16, 7583-7600 [3] Rasched, I. and Oberer, E. (1986) Microbiol. Rev. 50, 401-427 ******************************************************************************* Any statements made by the author are not meant to advocate the use of a particular commercial product or endorse any company. All opinions are those of the author and do not reflect the opinion of the National Cancer Institute or the National Institutes of Health. Copyright: This manuscript is not copyrighted by Elsevier Publishing Company. However, you may not reproduce any portion for resale or edit the text for redistribution, sale, or otherwise without written permission from the author. You found this at the World Wide Web (WWW) Uniform Resource Locator (URL) ftp://ftp.ncifcrf.gov/pub/methods/TIBS/mar94.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1994. Methods and reagents - Ghost plasmid of pBluescript Trends in Biochemical Sciences 19(3):139-140. ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* ******************************************************************************* ******************************************************************************* Note added in Press... Phage library contamination update In a recent issue of TIBS (18, 484-485), a story appeared in this column that outlined problems encountered with a genomic library that had seemingly been contaminated with bacteriophage T1. Recently, the company involved (Clontech) announced that they have identified the component responsible for the contamination as the in vitro lambda packaging extract used for the construction of the library, which they received from another supplier. A few libraries that showed the abnormally large plaques have since been reconstructed using an uncontaminated extract from a different supplier. We applaud Clontech's swift response upon learning of this problem. TIBS would like to thank Paul Hengen for bringing this contamination to light in his Methods and reagents column. If anyone has similar stories about molecular biological reagents, kits or equipment not living up to the promised standards, TIBS would like to hear from you. Please contact: The Editor, Trends in Biochemical Sciences, email: TIBS@PHX.CAM.AC.UK Phone: [44] 223 315961 *******************************************************************************