Difference between revisions of "Part:BBa K1641023"

Latest revision as of 01:38, 19 September 2015

Reporter of Invertase activity of Cre, pInv-rep-101LoxM

This is a reporter of invertase activity of Cre for Real-time measurement of dynamics of timing modules by SYSU-CHINA 2015.

The target sequence LoxP of Cre locates in the pInv-rep, surrounding a mcherry gene which is yet upside-down and transcribed by a constructive promoter. This mcherry-coding sequence can be inverted and restored to 5’ – 3’ direction at the existence of Cre or its EGFP fusion, rendering red signal. By real-time measurement of EGFP and mcherry, we can obtain data of Cre dynamics and simulate this process through modelling.

For this reporter: RTS type: LoxP mcherry type: BBa_J06504-BBa_M0052, which is mcherry-ssra(moderately fast) Promoter type: BBa_J23101

The functionality and measurement of this part

We used a real-time invertase dynamics testing system to understand the dynamics pattern of Cre. (See detail at [http://2015.igem.org/Team:SYSU_CHINA/Result Results of SYSU-CHINA]) 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.

This pInv-rep with promoter BBa_J23101 is a most commonly used reporter, which is the second strong promoter in our system.

Sequence and Features