Difference between revisions of "Part:BBa K1179058:Experience"

Latest revision as of 17:16, 18 September 2015

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Applications of BBa_K1179058

Cre is one of the most commonly used recombinase. For most situations it is used as integrase to add-in or knock-out some sequence. SYSU-CHINA in 2015 made full use of Cre as an invertase (the activity to invert a sequence between two RTS) to construct Micro-timer, a device providing timing function and rhythm to E. coli and yeast.

We used a real-time invertase dynamics testing system to understand the dynamics pattern of Cre. (See detail at Results of SYSU-CHINA超级链接) Briefly, for Cre-related timing module, a pair of LoxP is located upstream and downstream of and “up-side-down” RBS::mcherry sequence, which is the basic structure of our reporter vector. This meaningless sequence will be restored at the existence of Cre, expressed by another vecter, pInv-gen.

We used this system to test the activity of Cre, and the result shows a significant positive activity of invertase. We further understood that Cre has some special properties. 1, An ssra-tag linked to the C-term of Cre leads to lower expression rate, but can work pretty good. 2. Fusion protein of Cre-EGFP maintains a certain level of activity, but EGFP-Cre fusion evidently lose the activity (at least to a serious degree), even through EGFP-Cre seems to be more stable and accumulate in the cell for an extremely high concentration. However, this can be restored by adding an ssra.

In sum, Cre works pretty good as an invertase, and we confirmed functionality of this brick.

User Reviews

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