Figure 1

Optimizing the selection of the transposable cassette insertion into the target gene. A model of a cloned gene into the vector pGEM-t is shown in panels A and B. Two possible insertion events of the EZTN transposon are depicted: one in the vector (A) and the other in the cloned gene (B). The oligonucleotides used for cloning and analysis are shown as arrows. Panel C shows a PCR analysis of bacterial isolates after in vitro transposition. Oligonucleotides A, B and EZTN-R were used to estimate the approximate location of the transposon insertion. Lanes 1,3 and 5 correspond to insertion events probably located in the vector, as shown in the diagram of panel A. In this case, a 2.5 Kb insert corresponding to the size of the cloned gene is expected. Lanes 2,4,6 and 7 indicate insertion events in different points along the cloned gene, a situation illustrated in panel B. Panel C (right side) also shows the expected sequence obtained from the PCR products of lanes 2,4,6 and 7 by using the EZTN-R primer. The specific gene sequence located after the end of the transposon will indicate the precise location of the insertion as well as the orientation of the transposon.