Skip to main content
. 2020 Oct 9;11:578056. doi: 10.3389/fmicb.2020.578056

FIGURE 2.

FIGURE 2

Data from the in vitro recombination assay. (A) Demonstration that the recombinant integration host factor (IHF) and integrase preparations did not contain nucleases. Purified recombinant IHF and IntΦ24B were incubated with pCR2.1-Φ24B-attP600 to monitor these protein preparations for possible co-purifying nucleases. Lanes: MW, HyperLadderTM 1 kb (Bioline); 1, 100 ng pCR2.1-Φ24B-attP600; 2 and 3, 100 ng pCR2.1-Φ24B-attP600 with 1 μg of IHF or 1 μg of IntΦ24B, respectively, incubated at 30°C for 1 h. No co-purifying nuclease activity was detected. (B,C) In vitro recombination assay results using crude protein extracts. (B) Results after incubation for 6 h at 37°C. (C) Results after incubation for 24 h at 37°C. Reaction composition by lane numbers: MW, HyperLadderTM 1 kb (Bioline); 1 and 4, supercoiled pCR2.1-Φ24B-attP600 only, without crude protein extract; 2 and 5, linearized pCR2.1-Φ24B-attB600 only, without crude protein extract; 3 and 6, linearized pCR2.1-Φ24B-attB600 and supercoiled pCR2.1-Φ24B-attP600 with 500 ng crude protein extract from TOP10 E. coli; 7, supercoiled pCR2.1-Φ24B-attP600 and linearized pCR2.1-Φ24B-attB600 only. Recombination was only detected in lanes 3 and 6 with the presence of a 9-kb band. (D) PCR assay of the recombination products. MW, HyperLadderTM 1 kb (Bioline); 1, positive control using M13 F/R primers with pCR2.1-attP600 as template; 2, attRL PCR fragment amplified using M13 F/R primers; 3, attRL PCR fragment amplified using attP150 F/attB R primers; 4, negative control using M13 F/R primers without template.