Part:BBa_K510000:Experience

In order to characterize the function of the miniTn7BB-Gm minitransposon, we performed a set of experiments aimed to (i) determine whether miniTn7BB-Gm can transpose efficientely in an enterobacterial host (E. coli DH5α) and a non-enterobacterial host (P. putida KT2440), (ii) measure the transposition frequency when miniTn7BB-Gm was delivered by electroporation, chemical transformation and mating, (iii) determine whether insertion occurs site-specifically at the chromosomal attTn7 site, and (iv) determine whether flipase expression in trans can excise the drug resistance marker in the chromosomal copy of miniTn7BB-Gm.

Transposition of miniTn7BB-Gm in P. putida

In order to demonstrate miniTn7BB-Gm transposition into the P. putida KT2440 chromosome, pUC18Sfi-miniTn7BB-Gm was transferred to this host by two different means: (i) co-electroporation with the helper plasmid pTNS2 [pTNS2 is not replicative in P. putida, but transiently produces the Tn7 transposition machinery ( [http://http://www.nature.com/nmeth/journal/v2/n6/abs/nmeth765.html Choi et al. (2005)])], and (ii) tetraparental mating, using DH5α/pUC18Sfi-miniTn7BB-Gm as the donor, DH5α/pRK2013 as helper to provide the Tra functions, DH5α/pTNS2 to provide the Tn7 transposase and P. putida KT2440 as the recipient. P. putida clones bearing miniTn7BB-Gm insertions were selected on LB plates supplemented with Gm (and chloramphenicol when mating was used). Viable counts were also performed in both experiments, and plasmid transformation efficiency was determined using the gentamycin resistant replicative plasmid pBBR1mcs-5 in the electroporation experiment. The results are shown in Table 1.

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