Team:Hong Kong-CUHK/Parts

From 2010.igem.org

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<p><b>Rci site-specific recombinase</b></p>
<p><b>Rci site-specific recombinase</b></p>
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<p>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.</p>
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<p>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.</p>
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<p>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.</p>
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<p>With the expression of this gene, the DNA sequence would be shuffled randomly according to the specific repeat sites recognized by the recombinase.</p>
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<p><a href="/Image:Mess_Recom.png" class="image" title="Image:Mess_Recom.png"><img alt="Image:Mess_Recom.png" src="/wiki/images/d/d5/Mess_Recom.png" width="582" height="239" border="0"></a>
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<p>This is a Wide type Rci recombinase.</p>
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</p>
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<b>Recombinase activity</b>
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<ul>
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<center>
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<table align="center" border="1" cellpadding="4" cellspacing="0" style="border:#c9c9c9 1px solid; margin: 1em 1em 1em 0; border-collapse: collapse;">
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<li>In the recombination activity study:</li>
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<tbody><tr>
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<th>Intramolecular inversion frequency
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</ul>
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</th><th>0.062
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</th></tr>
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<ul>
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<tr>
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<th>Intramolecular deletion frequency
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<li>Intramolecular inversion frequency = 0.062</li>
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</th><th>0
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</th></tr>
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</ul>
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<tr>
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<th>Intramolecular recombination
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<ul>
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</th><th>0
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</th></tr></tbody></table>
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<li>Intramolecular deletion frequency = 0 (recombinase inactive on this function)</li>
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</center>
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<p><br />
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</ul>
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The inversion frequency is affected by the length of flanking DNA sequence in vivo. Please refer to <a href="/Part:BBa_K361001" title="Part:BBa K361001"><b>BBa_K361001</b></a>  
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</p>  
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<ul>
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<a name="Usage_and_Biology"></a><h3><span class="editsection">[<a href="/wiki/index.php?title=Part:BBa_K361000&amp;action=edit&amp;section=1" title="Edit section: Usage and Biology">edit</a>]</span> <span class="mw-headline">Usage and Biology</span></h3>  
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<p>Usage:
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<li>Intermolecular recombination = 0 (recombinase inactive on this function)</li>
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</p>  
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<ul><li>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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</ul>
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</li></ul>  
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<ul><li>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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<p>The inversion frequency is affected by the length of DNA sequence being flanked in vivo. Please refer to&nbsp;<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K361001">BBa_K361001</a></p>
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</li></ul>  
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<p><br />
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<p> </p>
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Biology:
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</p>  
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<p><strong>Usage and Biology</strong></p>
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<ul><li>In vitro study of conditions affecting recombination:
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</li></ul>  
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<p>Usage:</p>
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<p>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.<br />
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<ul>
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2. The inversion was sensitive to KCl concentration
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→Maximal inversion activity at a KCl concentration of 80 mM.<br />
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<li>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.</li>
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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 <sup>o</sup>C and 30 <sup>o</sup>C.<br />
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</ul>
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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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</p>  
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<ul>
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<ul><li>In vitro study of recombination:<br />  
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</li></ul>  
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<li>The Rci site-specific recombinase is now exploited to randomly rearrange the DNA sequence carrying data. It allows data encryption to be possible.</li>
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<p>About 15-20&nbsp;% inversion / recombination
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</p><p><br />  
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</ul>
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</p>
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<ul><li>Reference:
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<p>Biology:</p>
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</li></ul>  
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<p>[1] Gyohda, A. &amp; Komano, T. (2000). Purification and Characterization of the R64 Shufflon-Specific Recombinase. Journal of Bacteriology, 182 (10), 2787-2792.
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<ul>
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</p><p>[2] Gyohda, A., Zhu, S., Furuya, N. &amp; 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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</p>
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<li>In vitro study of conditions affecting recombination:</li>
+
-
 
+
-
</ul>
+
-
 
+
-
<p>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.</p>
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<ul>
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+
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<li>In vitro study of recombination:</li>
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+
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</ul>
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<p>About 15-20&nbsp;% inversion / recombination</p>
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Reference:<br />  
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<ul>
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</ul>
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<p>Gyohda, A. &amp; Komano, T. (2000). <em>Purification and Characterization of the R64 Shufflon-Specific Recombinase</em>. Journal of Bacteriology, 182 (10), 2787-2792.</p>
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-
 
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<p>Gyohda, A., Zhu, S., Furuya, N. &amp; Komano, T. (2005).<em> Asymmetry of Shufflon-specific Recombination Sites in Plasmid R65 Inhibits Recombination between Direct sfx Sequences</em>. The Journal of Biological Chemistry, 281 (30), 20772-20779.</p>
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<p> </p>
<p> </p>

Revision as of 02:58, 28 October 2010

Parts submitted

Parts

Part

Name

Type

BBa_K361000

Rci site-specific recombinase

Coding

BBa_K361001

Message gene template

DNA

BBa_K361011

Rci recombinase system

Generator

BBa_S04509

R0010:B0034 (lac promotor + RBS)

Intermediate

BBa_S04524

K361000:B0014 (Rci + terminator)

Intermediate

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.

Image: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

[edit] 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.

Message gene template

This message system consists of a unique designated message sequence, being inserted with specific repeat sequence for recognition site by Rci site-specific recombinase.

  • This message has 70 characters:

"We must learn to live together as brothers or perish together as tools"

  • In a 4 bits system, the total sequence is 280 bp.

In theory, the sequence with length equal to/ lower than 536 bp has high inversion frequency after 50 generation.

Reference:

Gyohda, A. & Funayama, N. (1997). Analysis of DNA Inversions in the Shufflon of Plasmid R64. American Society of Microbiology, 179 (6), 1867-1871.

Rci recombinase system

This is the protein genertors of Rci site-specific recombinase BBa_K361000, which is being linked with a promotor and a ribosome binding site in front of it, a double terminator after it.

Please refer to part   “Rci site-specific recombinase”

R0010:B0034 (lac promotor + RBS):

Construction intermediate of lac promotor and RBS (defined effiency =1)

K361000:B0014 (Rci + terminator)

Construction intermediate of BBa_K361000 and BBa_B0014 double bidirectional terminator

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