In view of the bimodal shape of the time course of Figure S1 (see Additional File 1) we picked 5 h as the most useful time for maximum conjugation/transposition LB-100 supplier events with a minimum of growth. The next step was to examine whether exconjugants had undergone authentic transposition events or they resulted from the

cointegration of pBAM1 into the host genome. 200 colonies were randomly selected and their sensitivity to the plasmid marker (ApR) tested. All 200 KmR clones turned out to be sensitive to the β-lactam antibiotic ampicillin (500 μg ml-1), thereby indicating that the insertion of the mini-transposon carried by pBAM1 had occurred as expected. In view of the high numbers, we click here wondered whether pBAM1 could also be delivered to P. putida cells in a suicide manner through electroporation instead of conjugation. Given that the plasmid cannot replicate in the recipient (see above) this method has the Lonafarnib solubility dmso potential advantage that every KmR colony developed on the selective plate must come from a unique transposition event and that siblings are then avoided. Table 1 shows that, despite being less efficient than conjugation, transformation of pBAM1 did result in a large number of KmR clones, in a dose-dependent fashion with regards to the amount of DNA entered in the transformation mixture. As before, all of 100 such KmR colonies tested were sensitive to Ap, as they

resulted from bona fide transpositions, rather than co-integration

of the donor plasmid into the target genome. Table 1 Transposition Inositol monophosphatase 1 frequencies of pBAM1   Resistance frequency Analyses of exconjugants Technique a Spontaneous b Non-spontaneous c Sample d Transposition e Cointegrates f Mating Not detectable 1.8 ± 0.53 × 10-3 200 200 0 Electroporation Not detectable 1.02 ± 0.38 × 10-7 100 100 0 a The pBAM1 plasmid was introduced into recipient cells either by five-hour tri-parental mating or by electroporation, letting the cells to recover after the electro-pulse in LB at 30°C for one hour. Electroporation figures are the average of the frequencies obtained using 100 ng (1.1 ± 0.5 × 10-7) and 500 ng (0.89 ± 0.2 × 10-7) of plasmid DNA. b Number of P. putida KT2440 colonies that acquire the marker resistance spontaneously, without mating or electroporation. c Total number of cells that acquired the mini-transposon, as measured by growth in kanamycin normalized to the total 3 × 107 donor cells. The 5 h mating frequency was averaged using a total of 16 independent experiments. Electroporation was referred to a final cell concentration of 6 × 1010 electrocompetent cells and the frequency determined with 6 independent experiments. d Number of independent colonies that were screened for the presence of the mini-transposon marker (kanamycin) and for the loss of the plasmid backbone marker (ampicillin). e Number of kanamycin resistant colonies.