Part:BBa_I718016:Experience

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UNIQabe1d88e5cbfb2f7-partinfo-00000000-QINU UNIQabe1d88e5cbfb2f7-partinfo-00000001-QINU

igem2010 UT-Tokyo

Our Cre recombinase assay device (CRAD), BBa_K313009, is a part originally designed to report the functionality of the Cre recombinase. We used this part as the lox66 site and expected that the double-terminator between the lox66 site and the lox771 site will be excised irreversibly when Cre is expressed, and GFP to be consequently expressed from the T7 promoter in the presence of the T7 RNA polymerase. We were intending to use GFP expressed in this way as a positive control to our important assay, the “terminator leakiness assay.”

However, contrary to our expectation, Rossetta (DE3) pLysS possessing CRAD and a Cre recombinase generator under the control of the T7 promoter, BBa_K313008 on its plasmids did not express GFP even after addition of IPTG. As a control we have checked in advance that Rossetta (DE3) pLysS possessing the GFP generator under the T7 promoter expresses GFP when IPTG is added.

We hypothesized that the cause of this unexpected failure is that the lox sites used in the CRAD assay device were not functional. The grounds on which we construct this hypothesis are that the sequence of this part lox66 and lox71, BBa_I718017, registered as parts were different to lox66 and lox71 sequences reported in some scientific journals. (e.g. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC137435/) Our sequencing showed that all sequences of the CRAD were in agreement to those reported in papers except for those of the lox66 and lox71 sites.

So, our next move was to evaluate the functionality of the lox66 and lox71 sequences registered as parts. Of course, there also remained the possibility that Cre or the host E.coli strain was the problem. Therefore we designed an in vitro assay with the use of purified Cre protein in order to eliminate these possibilities. We added the purified Cre proteins to purified plasmids possessing CRAD, or to purified plasmids possessing lox sequences as reported in scientific journals. In the latter experiment, the sequence between two lox sites was excised, but in the former experiment, the sequence between lox66 and lox71 sites was not excised.

In conclusion, our hypothesis that lox66 and lox71 registered as parts are dysfunctional seems to be valid. As expected based on the discrepancy in the sequences, the CRAD possessing the lox sites from the registry did not express GFP in the presence of Cre. We therefore propose that these two lox sites should be replaced.