Difference between revisions of "Part:BBa K801100"

 
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<partinfo>BBa_K801100 short</partinfo>
 
<partinfo>BBa_K801100 short</partinfo>
  
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This is an improved version of the RFP generating device <partinfo>BBa_J04450</partinfo> designed by [http://openwetware.org/wiki/Davidson:Davidson_2005  Team Davidson 2005] that is now RFC10 and RFC25 compatible.<br>
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In 2012 the [https://parts.igem.org Registry of Standard Biological Parts] defined <partinfo>BBa_J04450</partinfo> as the standard shipping part that is required for submission of backbones.<br>
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K801100 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K801100 SequenceAndFeatures</partinfo>
  
  
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===Functional Parameters===
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<partinfo>BBa_K801100 parameters</partinfo>
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The colonies are clearly red in color under natural light after about 18 hours.
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Smaller colonies are visibly red under UV. The RFP part does not contain a degradation tag and the RBS is strong.
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* LacI sensitive
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* CAP sensitive
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This part is commonly used, but can fail if the system contains LacI or CAP protein.  <br>
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(<small>--[[User:Meagan|Meagan]] 15:39, 23 July 2009 (UTC)</small>)
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<gallery>
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Image:J04450 - lightbox.jpg|BBa_J04450 visualized under non-UV lightbox
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Image:J04450 - UV 254nm.jpg|BBa_J04450 visualized under 254nm wavelength UV lightbox
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Image:J04500-colonies.jpg|BBa_J04450 Colonies
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</gallery>
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==Usage as a cloning tool==
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[http://2010.igem.org/Team:Groningen Team Groningen 2010] reports the usage of this part as a cloning tool. When ligating any part, or part assembly, into any standard backbone that contains this part, the non-restricted and single-restricted backbones that self-circularize will produce red colonies on rich media plates (we use TY). These undesired transformants can than be avoided in the screening for the correct construct. With this method, the backbone desired for a new construct does not need to be purified from agarose gel to decrease the amount of undesired tranformants caused by ligation of the original part present in the backbone.
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The amount of incorrect transformants depends, of course, on the ratio of backbone (mixed with J04450) vs. BioBrick insert, the size of the BioBrick insert, and whether the insert is an assembly of two BioBricks. The images below show two ligations with different efficiencies. 
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<gallery>
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Image:co-inef-gn.jpg| An inefficient assembly ligation of two BioBricks into the pSB1C3 backbone producing many red colonies.
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Image:co-ef-gn.jpg| A more efficient, single BioBrick ligation into the pSB1C3 backbone.
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</gallery>
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==Extension of the standard compability to RFC10 and RFC25==
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[http://2012.igem.org/Team:TU_Munich Team TU_Munich 2012] extended the standard compability of the RFP coding device (<partinfo>BBa_J04450</partinfo>) to RFC10 and [http://hdl.handle.net/1721.1/45140 RFC25] by adding the NgoMIV and AgeI restriction sites into the prefix and suffix of this part.Additionally two AgeI restriction sites that were present in the genrator itself were deletd. <br><br>
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This part may be used as a stndard insert for RFC10 as well as [http://hdl.handle.net/1721.1/45140 RFC25] backbones. This improvement became necessary because insertion of <partinfo>BBa_J04450</partinfo> into a RFC25 compatible backbone leads to the deletion of the desired [http://hdl.handle.net/1721.1/45140 RFC25] restriction sites that are needed for protein fusions.

Revision as of 14:53, 21 October 2012

RFP Coding Device - RFC10 and RFC25 compatible

This is an improved version of the RFP generating device BBa_J04450 designed by [http://openwetware.org/wiki/Davidson:Davidson_2005 Team Davidson 2005] that is now RFC10 and RFC25 compatible.

In 2012 the Registry of Standard Biological Parts defined BBa_J04450 as the standard shipping part that is required for submission of backbones.


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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]




The colonies are clearly red in color under natural light after about 18 hours. Smaller colonies are visibly red under UV. The RFP part does not contain a degradation tag and the RBS is strong.

  • LacI sensitive
  • CAP sensitive

This part is commonly used, but can fail if the system contains LacI or CAP protein.
(--Meagan 15:39, 23 July 2009 (UTC))

  • BBa_J04450 visualized under non-UV lightbox

  • BBa_J04450 visualized under 254nm wavelength UV lightbox

  • BBa_J04450 Colonies


Usage as a cloning tool

[http://2010.igem.org/Team:Groningen Team Groningen 2010] reports the usage of this part as a cloning tool. When ligating any part, or part assembly, into any standard backbone that contains this part, the non-restricted and single-restricted backbones that self-circularize will produce red colonies on rich media plates (we use TY). These undesired transformants can than be avoided in the screening for the correct construct. With this method, the backbone desired for a new construct does not need to be purified from agarose gel to decrease the amount of undesired tranformants caused by ligation of the original part present in the backbone. The amount of incorrect transformants depends, of course, on the ratio of backbone (mixed with J04450) vs. BioBrick insert, the size of the BioBrick insert, and whether the insert is an assembly of two BioBricks. The images below show two ligations with different efficiencies.


  • An inefficient assembly ligation of two BioBricks into the pSB1C3 backbone producing many red colonies.

  • A more efficient, single BioBrick ligation into the pSB1C3 backbone.

Extension of the standard compability to RFC10 and RFC25

[http://2012.igem.org/Team:TU_Munich Team TU_Munich 2012] extended the standard compability of the RFP coding device (BBa_J04450) to RFC10 and [http://hdl.handle.net/1721.1/45140 RFC25] by adding the NgoMIV and AgeI restriction sites into the prefix and suffix of this part.Additionally two AgeI restriction sites that were present in the genrator itself were deletd.

This part may be used as a stndard insert for RFC10 as well as [http://hdl.handle.net/1721.1/45140 RFC25] backbones. This improvement became necessary because insertion of BBa_J04450 into a RFC25 compatible backbone leads to the deletion of the desired [http://hdl.handle.net/1721.1/45140 RFC25] restriction sites that are needed for protein fusions.