Difference between revisions of "Part:BBa K361000"
 
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[[Image:Example.jpg]]__NOTOC__
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__NOTOC__
 
<partinfo>BBa_K361000 short</partinfo>
 
<partinfo>BBa_K361000 short</partinfo>
  
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This part will prouce a 384-amino acid Rci site-specific recombinase used in shufflon system, which is a site-specific DNA recombination between two specific repeat sites as recognition.
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This part is responsible for the functioning protein - Rci site-specific recombinase (384-amino acid) in Shufflon system, a site specific DNA recombination would occur between two specific repeat sites.
  
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After this recombinase is being expressed, it can alter the DNA sequence being inserted randomly with the specific repeat sites originally to become an unknown sequence.
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With the expression of this gene, the DNA sequence would be shuffled randomly according to the specific repeat sites recognized by the recombinase.
  
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[[Image:Mess_Recom.png]]
  
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[[Image:Recom.png]]
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'''Recombinase activity'''<center>
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{|{{Table}}
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!Intramolecular inversion frequency
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!0.062
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|-
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!Intramolecular deletion frequency
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!0
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|-
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!Intramolecular recombination
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!0
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|}
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</center>
  
  
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The inversion frequency is affected by the length of flanking DNA sequence in vivo. Please refer to [[Part:BBa_K361001|'''BBa_K361001''']]
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This is a Wide type Rci recombinase.
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*In the recombination activity study:
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*Intramolecular inversion frequency = 0.062
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*Intramolecular deletion frequency = 0 (recombinase inactive on this function)
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*Intermolecular recombination = 0 (recombinase inactive on this function)
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The inversion frequency is affected by the length of DNA sequence being flanked in vivo. Please refer to <partinfo>BBa_K361001</partinfo>
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===Usage and Biology===
 
===Usage and Biology===
 
Usage:
 
Usage:
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*The Rci site-specific recombinase is used for the bacteria to adapt the change in environment by rearrange their gene pattern to create a new species of bacteria that has selective advantage in that changed environment for survival.  
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*The Rci site-specific recombinase is utilized by the bacteria in order to adapt the environmental changes by rearranging their gene pattern, the DNA shuffling effect may provide selective advantages in the new environment to survive.  
  
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*The Rci site-specific recombinase is now exploited to randomly rearrange the DNA sequence carrying data. It allows data encryption to be possible.
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*Integrated into our system, the Rci site-specific recombinase now functions as an encryption system - shuffling information that are stored in DNA.
  
  
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*In vitro study of conditions affecting recombination:
 
*In vitro study of conditions affecting recombination:
 
1. Additions of magnesium ion, EDTA, or ATP had little effect on recombination
 
1. Additions of magnesium ion, EDTA, or ATP had little effect on recombination
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→Neither an energy source nor divalent cations are necessary.
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→Neither an energy source nor divalent cations are necessary.<br>
 
2. The inversion was sensitive to KCl concentration
 
2. The inversion was sensitive to KCl concentration
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→Maximal inversion activity at a KCl concentration of 80 mM.  
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→Maximal inversion activity at a KCl concentration of 80 mM.<br>
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3. The inversion reaction showed a broad pH optimum between 7.6 and 8.5.
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3. The inversion reaction showed a broad pH optimum between 7.6 and 8.5.<br>
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4. The inversion reaction has a higher reaction activity at 42 oC as compared with that of 25 oC and 30 oC.
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4. The inversion reaction has a higher reaction activity at 42 oC as compared with that of 25 <sup>o</sup>C and 30 <sup>o</sup>C.<br>
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5. Negatively supercoiled DNA substrate was required for Rci protein. Little to zero recombination was observed in relaxed or linear DNA.  
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5. Negatively supercoiled DNA substrate was required for Rci protein. Little to zero recombination was observed in relaxed or linear DNA. <br>
  
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*In vitro study of recombination:
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*In vitro study of recombination:<br>
 
About 15-20 % inversion / recombination
 
About 15-20 % inversion / recombination
  
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*Reference:
 
*Reference:
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Gyohda, A. & Komano, T. (2000). Purification and Characterization of the R64 Shufflon-Specific Recombinase. Journal of Bacteriology, 182 (10), 2787-2792.
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[1] Gyohda, A. & Komano, T. (2000). Purification and Characterization of the R64 Shufflon-Specific Recombinase. Journal of Bacteriology, 182 (10), 2787-2792.
  
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Gyohda, A., Zhu, S., Furuya, N. & Komano, T. (2005). Asymmetry of Shufflon-specific Recombination Sites in Plasmid R65 Inhibits Recombination between Direct sfx Sequences. The Journal of Biological Chemistry, 281 (30), 20772-20779.
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[2] Gyohda, A., Zhu, S., Furuya, N. & Komano, T. (2005). Asymmetry of Shufflon-specific Recombination Sites in Plasmid R65 Inhibits Recombination between Direct sfx Sequences. The Journal of Biological Chemistry, 281 (30), 20772-20779.
  
 
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Latest revision as of 22:37, 27 October 2010

Rci site-specific recombinase

This part is responsible for the functioning protein - Rci site-specific recombinase (384-amino acid) in Shufflon system, a site specific DNA recombination would occur between two specific repeat sites.

With the expression of this gene, the DNA sequence would be shuffled randomly according to the specific repeat sites recognized by the recombinase.

Mess Recom.png

Recombinase activity
Intramolecular inversion frequency 0.062
Intramolecular deletion frequency 0
Intramolecular recombination 0


The inversion frequency is affected by the length of flanking DNA sequence in vivo. Please refer to BBa_K361001

Usage and Biology

Usage:

  • The Rci site-specific recombinase is utilized by the bacteria in order to adapt the environmental changes by rearranging their gene pattern, the DNA shuffling effect may provide selective advantages in the new environment to survive.
  • Integrated into our system, the Rci site-specific recombinase now functions as an encryption system - shuffling information that are stored in DNA.


Biology:

  • In vitro study of conditions affecting recombination:

1. Additions of magnesium ion, EDTA, or ATP had little effect on recombination →Neither an energy source nor divalent cations are necessary.
2. The inversion was sensitive to KCl concentration →Maximal inversion activity at a KCl concentration of 80 mM.
3. The inversion reaction showed a broad pH optimum between 7.6 and 8.5.
4. The inversion reaction has a higher reaction activity at 42 oC as compared with that of 25 oC and 30 oC.
5. Negatively supercoiled DNA substrate was required for Rci protein. Little to zero recombination was observed in relaxed or linear DNA.

  • In vitro study of recombination:

About 15-20 % inversion / recombination


  • Reference:

[1] Gyohda, A. & Komano, T. (2000). Purification and Characterization of the R64 Shufflon-Specific Recombinase. Journal of Bacteriology, 182 (10), 2787-2792.

[2] Gyohda, A., Zhu, S., Furuya, N. & Komano, T. (2005). Asymmetry of Shufflon-specific Recombination Sites in Plasmid R65 Inhibits Recombination between Direct sfx Sequences. The Journal of Biological Chemistry, 281 (30), 20772-20779.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 362
    Illegal NgoMIV site found at 857
  • 1000
    COMPATIBLE WITH RFC[1000]