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.