Restriction digests of the clone give the following sizes (kb): EcoRI--5.4, 3.3; BglII--8.8; HindIII--8.8. Cloning sites are positioned in the EcoRI endonuclease gene, regulated by a lambda promoter. In E. coli hosts providing the temperature-sensitive lambda repressor cI857, growth at 37C will occur only if the endonuclease gene is interrupted. When using the positive selection feature, the E. coli recipient should not be a lambda lysogen because the feature is turned off by a lambda repressor. The non-enzymatic chloramphenicol resistance gene, derived from Rhodococcus rhodochrous SC2318 (ATCC 12674), does not confer resistance in E. coli. The plasmid confers immunity to nocardiophage Q4. Utilization of the positive selection feature of this vector requires growth (37C, 50 ug/ml ampicillin) in hosts not expressing cI857, before transformation into nocardioforms. Subsequent large-scale growth of this stock (100 ug/ml ampicillin, 30 ug/ml rifampicin), allows preparation of enough plasmid DNA to transform the nocardioform recipient. Libraries should be allowed to grow in nocardioforms at 26 degrees for 12 hours before introducing selection. Chloramphenicol should be applied underneath the agar and be allowed to diffuse to a concentration of 40 ug/ml. Shuttle vector permitting positive selection in E. coli. E. coli MM294-1(lambda) is a rifampicin resistant derivative of E. coli MM294. The order of the major features in the plasmid is: AccIII - phage Q4 replicon - StuI - Cm-R gene - BbrPI - BbrPI - AccIII - EcoRI gene - ampR. |
