Difference between revisions of "Part:BBa K1641024"
 
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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.
 
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.
  
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For this reporter:  
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For this reporter:
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RTS type: LoxP;
 
RTS type: LoxP;
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+
 
 
mcherry type: BBa_J06504-BBa_M0052, which is mcherry-ssra(moderately fast);
 
mcherry type: BBa_J06504-BBa_M0052, which is mcherry-ssra(moderately fast);
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+
 
 
Promoter type: BBa_J23100;
 
Promoter type: BBa_J23100;
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----
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'''The functionality and measurement of this part'''
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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.
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[[File:SYSU-repstruct.jpg]]
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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.
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[[File:SYSU-reptable.jpg]]
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[[File:SYSU_result gprh.jpg]]
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This pInv-rep with promoter BBa_J23100 is most strong promoter in our system.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 01:28, 19 September 2015

Reporter of Invertase activity of Cre, pInv-rep-100LoxM


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_J23100;


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.


SYSU-repstruct.jpg


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.


SYSU-reptable.jpg

SYSU result gprh.jpg


This pInv-rep with promoter BBa_J23100 is most strong 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]