Difference between revisions of "Part:BBa K1179058"
 
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It encodes for Cre recombinase, which when present with LoxP sites performs Cre-Lox recombination and creates a Holliday junction.
 
It encodes for Cre recombinase, which when present with LoxP sites performs Cre-Lox recombination and creates a Holliday junction.
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----
  
 
Cre is one of the best understood Tyr-family recombinases. Although it is originally a integrase utilized by phages to infect its host. It is now one of the most commonly used tool for DNA knocking-out or insertion. If the two Lox sites are parallel, a deletion of sequence between them will happen. However, if the two loxP are anti-parallel, the sequence will be inverted into up-side-down. Such kind of activity is called invertase activity.
 
Cre is one of the best understood Tyr-family recombinases. Although it is originally a integrase utilized by phages to infect its host. It is now one of the most commonly used tool for DNA knocking-out or insertion. If the two Lox sites are parallel, a deletion of sequence between them will happen. However, if the two loxP are anti-parallel, the sequence will be inverted into up-side-down. Such kind of activity is called invertase activity.
 +
 +
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. Before we use Cre to construct complicate pathway, we used a real-time invertase dynamics testing system to understand the dynamics pattern of Cre. 
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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.
 +
 +
One of the typical results of our activity testing experiments is like following picture. When inducer is added into the culture, Cre-EGFP start expression, which overturns the reporter sequence, and red signal is generated at an increasing speed.
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[[File:typical.jpg]]
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Please see Experience page or [http://2015.igem.org/Team:SYSU_CHINA/Result Wiki of SYSU-CHINA] for more information.
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In sum, The invertase activity of recombinase Cre has been confirmed, and we discovered some interesting properties of Cre.
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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 18:38, 18 September 2015

Cre entry vector

This part is submitted under the RFC 65 standard.

It encodes for Cre recombinase, which when present with LoxP sites performs Cre-Lox recombination and creates a Holliday junction.

Cre is one of the best understood Tyr-family recombinases. Although it is originally a integrase utilized by phages to infect its host. It is now one of the most commonly used tool for DNA knocking-out or insertion. If the two Lox sites are parallel, a deletion of sequence between them will happen. However, if the two loxP are anti-parallel, the sequence will be inverted into up-side-down. Such kind of activity is called invertase activity.

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. Before we use Cre to construct complicate pathway, we used a real-time invertase dynamics testing system to understand the dynamics pattern of Cre.

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.

One of the typical results of our activity testing experiments is like following picture. When inducer is added into the culture, Cre-EGFP start expression, which overturns the reporter sequence, and red signal is generated at an increasing speed.


Typical.jpg


Please see Experience page or [http://2015.igem.org/Team:SYSU_CHINA/Result Wiki of SYSU-CHINA] for more information.

In sum, The invertase activity of recombinase Cre has been confirmed, and we discovered some interesting properties of Cre.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 512
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 198
  • 1000
    COMPATIBLE WITH RFC[1000]