Restriction digests of the clone give the following sizes (kb): AvaI--3.0, 1.3, 1.2; BamHI--5.4; EcoRI--2.95, 2.6. Cultures should be grown in minimal media for more reproducible induction of expression. Expression is induced in glycerol-containing media by addition of arabinose. Expression is repressed by addition of glucose or other catabolites. One of several tightly controlled expression vectors (ATCC 87393-87402) regulated by the arabinose operon. The vectors differ in replicon, antibiotic resistance marker, multiple cloning site and mechanism of initiation of translation. The following primers can be used for sequencing of cloned inserts: 5' primer (27 - 8 bp upstream of the NheI site) 5'-CTGTTTCTCCATACCCGTT-3'; and one of two 3' primers: 3' primer 1 (2 - 19 bp downstream of the HindIII site) 5'-CTCATCCGCCAAAACAG-3'; 3' primer 2 (17 - 33 bp downstream of the HindIII site) 5'-GGCTGAAAATCTTCTCT-3'. Plasmid copy number is low due to the p15A replicon. This vector can be used when reduced gene expression is desirable. Plasmid contains bla (ampR) sequences following the rrnB terminator which could promoter recombination if this plasmid is used in combination with other compatible ampR plasmids. Plasmid is compatible with pBR-derived plasmids and may be used for coexpression of cloned inserts. Recombination can be avoided by the use of recA host strains, or it can be used to advantage to intentionally exchange markers among plasmids. |
