Part:BBa_K1641035

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

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, which is mcherry but without ssra;

Promoter type: BBa_J23101;

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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 low-intensity promoter in our system.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]