Part:BBa_K1641026
Reporter of Invertase activity of Cre, pInv-rep-110LoxM
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_J23110;
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_J23106 is a medium-intensity promoter in our system.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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