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BAC Library Construction

 Vector pBACe3.6 Information/Map    

In the course of our initial work with PAC libraries and PAC vectors, we obtained feedback from several sequencing centers who considered the costs of sequencing PAC versus BAC clones.  A disadvantage was the size of the PAC vector (16 kb left in recombinants) as compared to the size of the BAC vector (7 to 8 kb, depending on the vector variant).  For the use of BACs versus PACs with inserts in the range of 100-150 kb, this means that a larger fraction of the clones in shotgun sequence libraries (M13 or pUC) represents the PAC/BAC vector sequence. This results in a few percent increase in the cost per base pair of sequence reads.

We have, therefore,improved the BAC vector (Shizuya H. et. al. 1992, Proc. Natl. Acad. Sci. USA 89:8794-8797) to include some of the retrofitting options included in our pPAC4 vector We also included the sacBII gene for use as a positive-selection marker to favor recombinant clones over non-insert background colonies. The sacBII gene from Bacillus amyloliquefaciens was derived from Nat Sternberg's P1 vector (Pierce et al. 1992, Proc. Natl. Acad. Sci. USA 89:2056-2060). The toxicity of the sacBII gene is a result of the conversion of fructose (derived from sacharose) into polyfructose (levan), which is toxic in E.coli.  The cloning vector and recombinant clones do NOT express the sacBII gene and hence are sucrose-resistant. Undesirable clones derived from the deleted cloning vector (without the "green" stuffer fragment) are sucrose-sensitive and ,hence, do not form colonies on sucrose-containing media.

The new vector has been named pBACe3.6.  Specifically, we maintained the wildtype loxP site, added an additional mutant loxP511 site, a site for the intron encoded nuclease PI-SceI and the Tn7att site. 

The presence of this pUC plasmid serves dual functions: high copy number of the vector for preparing large quantities and appropriate disruption of the sacBII gene to increase viability of the vector containing strain.  In addition to the BamHI site, 5 additional sites can be used for preparing BAC libraries: SacI, SacII, MluI, EcoRI, and AvaIII.  The EcoRI site is particularly important in view of the use of this site in the RARE cleavage procedure, thus opening the possibility for selective cloning of similar fragments from different DNA donors.

Reference: Frengen, E. et al. (1999) A Modular, Positive Selection Bacterial Artificial Chromosome Vector with Multiple Cloning Sites. Genomics 58: 250-253. Reprints availiable upon request.

pBACe3.6 clones have chloramphenicol antibiotic resistance.  Clones should be grown in LB containing 12.5 ug chloramphenicol/ml.

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