Part:BBa_K1179058

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.

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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.

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.

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]