Restriction digests of the clone give the following sizes (kb): EcoRI--10.5; BamHI--10.5; ClaI--10.5. Insert expression is regulated by the Mu middle promoter that is induced by Mu replication and that depends on the transactivator mor (middle operon regulator). The mor gene is expressed from vector sequences by readthrough from the nptII promoter. Induction of replication may increase copy number up to 50-fold. For plasmid replication, Mu lysogens such as E. coli MH3870 are recommended. This host cannot package phage because it has an amber mutation (Cam4005) in the C gene. Transposition-competent, packaging-defective phage can be produced by transformation of the plasmid into a Mu lysogen such as E. coli MC1040 that provides packaging function. For stable expression, kanamycin resistant, ampicillin sensitive colonies should be identified from non-lysogenic hosts such as E. coli MH4997 transduced with recombinant, defective phage. After integration, the vector is stable without antibiotic selection. Mu lysates containing cloned inserts can be used to infect other bacteria such as Serratia, Erwinia and Citrobacter. Chromosome-based (integrating) expression vector based on bacteriophage Mu. Growth at 42C inactivates the temperature sensitive Mu repressor (cts) and allows expression of the insert as well as genes controlling replication and integration of phage Mu. The order of the major features in the plasmid is: attL - Mu repressor (cts62) - HindIII - ner - A gene - B gene - XhoI/Tn5L - HindIII/kanR/XhoI - mor - middle promoter - T7 enhancer g10L/XbaI - MCS(EcoRI/ClaI/BamHI) - attR - ampR/pMB1 ori. |
