Construction of pTetFlp

Strategy for deletion of Rha-RedRecA and dhfr

As shown above the kan.gcc which encodes for kanamycin resistance was amplified from the Pirate-km.gcs plasmid (from Stewart lab). This Kan gene has homology arms to enable its introduction into the pRedFlp plasmid (from Stewart lab) by Red/ET recombination. The pTetFlp KanR has a pSC101 temperature sensitive origin (grows at 30°C but not at 37°C). The PCR product was isolated and purified from the gel. The gel was as shown below



Red/ET was done to introduce the KanR gene into the pTetFlp. The resulting plasmid was named pTetFlp KanR. This was digested with PstI, and showed the following results



From the 14 minis, 3 correct clones were re-electroporated, in order to purify it. This is a high copy plasmid and the first step of Red/Et mostly results in a mixture of the original plasmid and the desired plasmid. Re-electroporation and transformation results in purer products. 16 minis were done, of which 13 were correct, as shown below



Strategy for insertion of Flp reporter



As shown in the strategy above, pTetFlp KanR was digested using XhoI in order to linearize it.

F3-ZeoR-F3-RFP (plasmid ordered from GeneArt), is as shown in Fig 3. This when digested using PvuII resulted in the fragment shown in Fig 4. It carries a zeocin resistance gene (ZeoR). The ZeoR gene is flanked by F3 sites that are similar to the FRT sites (vary in a few bases) and is hence recognized and excised by Flippase (Flp). There is a terminator upstream of the second F3 site and a RFP gene is immediately downstream. So in the absence of Flp RFP is not expressed, But when Flp excises the region between the F3 sites, RFP is expressed.





The PvuII digest was as shown below. From this the band of size 1549 bp was isolated from the gel and purified.



Both pTetFlp KanR and F3-ZeoR-F3-RFP fragments were linear, so linear+linear Red/ET was done. The digets of 13 mini preps were as follows.



3 correct clones were re-electroporated and transformed. 23 minis were done from this. This is shown below.