Restriction digests of the clone give the following sizes (kb): KpnI--7.2, 2.1; XhoI--8.2, 1.2; ClaI--9.0, 0.4. Integration can be achieved by deletion of the KpnI fragment of the vector (containing the origin of replication and ampicillin resistance gene), followed by transformation of a tyrA deficient host and selection for tyrosine prototrophy. A host lacking pheA-tyrA-aroF is recommended. Integration efficiency can be improved by co-infection of the transformed cells with an integration-deficient helper phage, such as lambda(imm434b104). After temperature induction at 42 C, the tyrA gene will be excised from the chromosome and lost, effectively stopping cell growth in tyrosine deficient media. Vector allowing insertion of the tyrA gene into the E. coli chromosome and controlled excision of the same DNA upon temperature induction. |
