Restriction digests of the clone give the following sizes (kb): EcoRI--4.6; HindIII--4.6; PstI--3.6, 1.1. 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'. Vector contains the Shine-Dalgarno box and 5 amino acids of the araB gene, but does not necessarily form fusion proteins with cloned inserts. Higher expression may still be achieved due to the second SD box despite out-of-frame cloning. Cloned inserts should provide a translation initiation sequence (ATG) and ribosome binding site for expression. |
