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Figure 2 | BMC Microbiology

Figure 2

From: Fast, easy and efficient: site-specific insertion of transgenes into Enterobacterial chromosomes using Tn7 without need for selection of the insertion event

Figure 2

Protocol for use of transgene insertion plasmid. 1) Transform the plasmid into the strain of choice and select transformants on LB + ampicillin at 32°C. Introduction of the plasmid by conjugation is also an option. 2) Streak once to ensure that the cells are carrying the plasmid, then grow non-selectively in LB at 32°C. In all strains tested thus far, leaky expression of TnsABCD during this step has been sufficient to allow site-specific transposition into the attTn7, but 0.1% arabinose may be added to increase levels of expression of TnsABCD. 3) Plate cells at 42°C to block replication of the plasmid. 4) Streak once at 42°C to ensure loss of the plasmid, then verify insertions in the attachment site by PCR.

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BMC Microbiology

ISSN: 1471-2180

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