Wild, J., Hradecna, Z. and Szybalski, W.: Derivatives of pBAC vectors allowing conditional amplification of inserts in E. coli hosts. Abstracts of Papers Presented at the 1997 Meeting on Molecular Genetics of Bacteria & Phages, Cold Spring Harbor, NY, August 25-30, 1998, page 136.
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DERIVATIVES OF pBAC VECTORS ALLOWING CONDITIONAL AMPLIFICATION
OF INSERTS IN E. coli HOSTS
Jadwiga Wild, Zdenka Hradecna and Waclaw Szybalski. McArdle Laboratory,
University of Wisconsin Medical School, Madison, WI 53706,
e-mail: szybalski@oncology.wisc.edu
The bacterial artificial chromosome (BAC) system [Shizuya et al., PNAS
89 (1992) 8794-8797] is widely employed for construction of stable genomic DNA
libraries. The pBAC vectors, based on the F plasmid replicon, allow stable maintenance
of large genomic DNA fragments as single-copy plasmids. However, the disadvantage
of these vectors is a low yield of both the vector and of the cloned DNA. To
overcome this limitation, we have re-engineered pBeloBAC11, using yeast and
bacterial plasmid elements, to allow conditional production of multiple copies
of the excised cloned fragment or of the entire vector, while retaining all
advantages of the single-copy vector.
We have cloned one FRT element at
the HpaI site of the pBeloBAC11, and the second FRT, together
with an oriV element, at the XhoI site; thus two FRTs were
flanking the MCS(multiple cloning site)-CmR-oriV
segment, resulting in the pBAC(FRT-oriV) vector. The two FRT sites
were parallel, so as to allow excision of intervening oriV-carrying DNA
upon supply of Flp. The principle of excision and amplification is analogous
to that described by Wild et al. [Gene 179(1996)181-188]. Furthermore, we constructed
two specific E. coli hosts. Host JW192 (DH5a-Rep)
supplies the TrfA function constitutively; this results in 25- to 50-fold amplification
of pBAC(FRT-oriV) to be used as the cloning vector. Host JW303 (DH10b-Flp/Rep)
carries in its genome the tetR-Ptet-FLP-FRT-trfA(inverted)-FRT
cassette [Sektas and Szybalski, Molec. Biotechnol. 9 (1998) 17-24] inserted
at the attB site. Genes FLP and trfA are not expressed
until the Ptet
promoter/operator is derepressed by adding the autoclaved chlortetracycline
(cTc). The Flp protein, synthesized only upon induction, exerts dual action.
(i) It inverts trfA, leading to the production of TrfA protein,
as directed by the Ptet promoter. (ii) It excises the
FRT-flanked DNA segment from pBAC(FRT-oriV).
Using JW303 and cTc induction, we have shown that cloned DNA fragments in the
range of 3 to 100 kb are rapidly excised, circularized and amplified about 25-
to 50-fold, whereas clones in uninduced host are stably maintained as a single
copy. The optimization of the excision-amplification process will be discussed,
including the optimal conditions for Flp and TrfA induction and the use of different
trfA alleles, as well as their combinations.