Team:HZAU-China/Characterization

Basic works

Confirmation of the unexpected recombination of lox71 site

Cre recombinase is the type I topoisomerase found in phage P1 that can catalyze site-specific recombination between loxP sites. Lox71 is a mutation of loxP which can also be specifically recognized by Cre recombinase. But the result of the sequencing in DH5α may suggest that our design cannot be used in E.coli because recombination will happened without cre recombinase. In order to confirm it, we constructed the confirmation device shown in the figure 3 below. Then we transformed the device to kinds of the competent E.coli cells (DH5α，DH10B，BL21（DE3）) we got from different lab.

Fig.3 Confirmation Device

After cultivate with IPTG in the 37°C shaking incubator at the rotational speed of 180 rpm/min for 24 hours, we used the fluorescence microscope to observe bacteria and you can see result at fig.4(DH5α) and fig.5(DH10B) below.

Fig.4 unexpected recombination in DH5alpha

Fig.5 unexpected recombination in DH10B

At the same time, the samples were sequenced and anything was OK.

In summary, we confirmed the unexpected recombination of lox71 site even if the device is in the stable E.coli strains such as the DH5α and DH10B. But the probability of its occurrence is extremely low. And it doesn’t affect the sequencing result in a short time. The tetR protein might be poisonous to the E.coli, when the promoter is deleted by lox71 site, the bacteria will get the survival advantage and quickly occupy the dominant position in LB medium. Therefore, we recommend that you save the plasmid rather than bacteria when you use the cre/lox system.