The Formaiion of pBHIO , plasmid vehical

The Formation of pBHIO :

   Digestion of pBR322 with Leo RI produces c linear duplex with short, singk-siranded tails. These tails were convened to duplcxcs by rcalmcnl with T4 DNA polymerasc to ich1 a blunt.cndcd mokculc, The cndoondcasc Hae III which produces blunt ends, was used to excise the lee control region from bactenophage >.piac. With the aid of T4 DNA ligase. which permits ligation of blunt-ended molecules, the be control region was joined to the blunt ended pBR322 densaxie. II should be noted that this Procedure

results us the formation of two Leo RI tiles, one on either side of the i.e control region

       After ligation, the inature was transIonned usto L. coil and selection made for blue colonies on medium containing ampicillin, tetracycline and the chromogenic substrate S-bromohioro4indoIyI-lD-galactosidc (Xgal). The raüonale for this was as foIios. Xgal is not an inducer of U-galacsoaidasc but is ckavcd by a•galactos.dase releasing a blue indolyl dcnvatisc. Since Xgal is not an inducer, only mutants conuiiulisre for

as.laciosdac produce blue colonies on medium containing Xgal. Plasmid 9BHIO is maintatned as a relaxed plasmid. i.e. multiple copes per ccU. Thus, cells carrying pBHIO have multiple copies 01’ the Inc control

region and can titrate out all the repressor produced by the single chromosomal lac i gene leading to a constitulive phenotype. Clearly there arc two possible orientations for the insertion of the Hat III fragment but these can be distinguished by the location of an asymetrically placed Hha site relative to the Hind Ill site