For preparing genomic DNA libraries;

1. Stable maintenance of inserted fragments in Escherichia coli

The TAC vector contains the P1 bacteriophage replicon, which maintains the vector in a single copy, and therefore renders foreign DNA fragments stable, in E. coli cells. The vector also contains the pRiA4 replicon of the Ri plasmid, which ensures a vector copy number of 1 in Agrobacterium tumefaciens. The kanamycin resistance marker gene (NPTI), modified by removal of the Hind III site, is included in the vector to allow selection of clones in both E. coli and A. tumefaciens by culture in the presence of kanamycin.

Unique Hind III and Bam HI cloning sites for the preparation of genomic DNA libraries are included in the TAC vector between the sacB gene and its promoter. The efficient ligation of Hind III cohesive ends ensures that large-scale genomic DNA libraries can be readily prepared with the use of the Hind III site. However, Hind III fragments of relatively small size may not encompass entire genomes. The use of the Bam HI site is thus recommended for preparation of another genomic DNA library from the same DNA source. The Bam HI site also may be used for preparing libraries with small (<30 kb) Sau 3AI-Mbo I fragments.

Insert-bearing clones can be selected on sucrose-containing agar plates. The insertion of a DNA fragment at the cloning sites between the sacB gene and its promoter reduces the production of levansucrase, which is encoded by the sacB gene, in E. coli. Given that the production of this enzyme is lethal for E. coli in the presence of 5% sucrose, culture of transformed cells in the presence of this sugar eliminates those harboring the empty vector.

1. Plasmid preparation from E. coli

The low-yield disadvantage for DNA preparation of P1 replicon-mediated maintenance of the TAC plasmid in a single copy in E. coli can be overcome by releasing suppression of the P1 lytic replicon with isopropyl-b-D-thiogalctopyranoside (IPTG). The resulting plasmid amplification ensures that several micrograms of TAC plasmid DNA can be obtained from a 3-ml culture grown in the presence of 0.5 mM IPTG (Protocol 1). (Note that 0.3 to 0.5 mM IPTG is sufficient for releasing suppression in E. coli strain DH10B.)

The TAC vector includes TAIL-PCR (thermal asymmetric interlaced polymerase chain reaction) primer sequences (R1, R2, R3, L1, L2, and L3) flanking the cloning sites (Fig. 1b). The TAIL-PCR protocol, which requires three specific primers for the vector sequence and an arbitrary degenerate primer, but not an insert-specific primer, has been applied successfully for the isolation of end fragments of inserted DNA from many TAC clones in our and other laboratories.

Reference for the TAIL-PCR protocol:
Liu, Y.-G. and Whittier, R.F. (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25, 674-681

For transfering large DNA fragments into plant genomes by Agrobacterium-mediated protocols

1. Cis elements for gene transfer

The TAC vector contains left (LB) and right (RB) borders, the cis sequences required for Agrobacterium-mediated gene transfer in plants. The plant-selectable marker gene HPT, which encodes hygromycin phosphotransferase, is also included in the vector under the control of the nopaline synthase gene (nos) promoter (Pnos) and upstream of the nos terminator (Tnos). The HPT construct is included together with the cloning sites and sacB between the LB and RB sequences.
TAC plasmids containing large (~100 kb) inserts are transferred efficiently into A. tumefaciens by electroporation (Protocol 1). The pRK2 oriT sequence, which is used for delivering plasmids from E. coli to A. tumefaciens by the triparental-mating method, is therefore not included in the TAC vector.

1. The I-SceI sites for cutting the whole insert fragment out

The TAC vector contains two I-Sce I sites, one upstream of the Pnos sequence and the other downstream of the cloning sites, to allow Southern blot analysis of the integrity of large transgenes. Thus, the entire insert sequence together with the HPT tag can be released from the plant genome by digestion with I-Sec I and be detected by Southern analysis with an HPT gene probe. Given that I-Sce I recognizes an 18-bp sequence, recognition sites for this enzyme should occur only once per 6.9 x 1010 bp of random DNA sequence. Most plants should therefore not contain a I-Sce I site. Protocols describing the preparation of high molecular weight genomic DNA and I-Sce I digestion for Southern analysis are included in Protocol 2.

Unique sites for Asc I, Sfi I, Srf I, Fse I, and Not I, each of which recognizes rare 8-bp sequences, are included in the TAC vector flanking the Hind III and Bam HI cloning sites. After a TAC clone containing a large genomic DNA fragment is shown to complement the mutation of interest, these sites can be used to create a series of deletion clones in order to identify the target gene by further complementation tests.