Difference between revisions of "Plasmid backbones/Assembly of protein fusions"
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Protein engineers often need to do in-frame assemblies in order to assemble together different signal sequences, proteins domains, and protein tags.  BioBrick&trade; standard assembly is not well-designed for this task because the scar sequence formed by the SpeI-XbaI ligation is 8 base pairs long, so assembly of protein domains causes frameshifts.  As a result, different groups have developed modified or alternative assembly schemes to facilitate assembly of protein domains.  
 
Protein engineers often need to do in-frame assemblies in order to assemble together different signal sequences, proteins domains, and protein tags.  BioBrick&trade; standard assembly is not well-designed for this task because the scar sequence formed by the SpeI-XbaI ligation is 8 base pairs long, so assembly of protein domains causes frameshifts.  As a result, different groups have developed modified or alternative assembly schemes to facilitate assembly of protein domains.  
  
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==Plasmid backbones that adhere to the Silver standard ([http://bbf.openwetware.org/RFC.html BBF RFC] 23)==
  
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==Plasmids that adhere to the Silver standard==
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[[Image:SilverFusionScheme.png|right]]
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{|
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|[[Image:CarolineAjoFranklingPhoto.jpg|60px|center]]
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|Caroline Ajo-Franklin developed the Silver lab protein fusion vectors [[Part:BBa_J63009|BBa_J63009]] and [[Part:BBa_J63010|BBa_J63010]] in Pam Silver's lab.
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Pam Silver's lab has developed the Silver standard for assembling protein domains.  It relies on shortening the BioBrick prefix and suffix each by 1 base pair such that the resulting SpeI-XbaI scar is only 6 base pairs long and protein domains can be assembled in frame. 
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As shown in the figure 3 (right), the Silver standard results in the inclusion of a Thr-Arg (ACT AGA) two amino acid junction between protein domains.
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One disadvantage of the Silver standard is that the codon AGA which encodes Arginine is a rare codon in ''E. coli''.  Rare codons can prevent over-expression of proteins in E. coli.
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===References===
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{{:Silver_standard/References}}
  
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==Plasmid backbones that adhere to the Freiburg standard ([http://bbf.openwetware.org/RFC.html BBF RFC] 25)==
  
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|Raik Gruenberg developed the protein fusion vectors [[Part:BBa_J18901|BBa_J18901]], [[Part:BBa_J18902|BBa_J18902]] and [[Part:BBa_J18903|BBa_J18903]] that adhere to the Freiburg standard.
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===References===
 
===References===
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'''A New BioBrick Assembly Strategy Designed for Facile Protein Engineering'''<br>
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{{:Assembly standard 25/References}}
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''MIT SBWG Technical Reports,'' 2006 Apr 20<br>
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Ira Philips, Pam Silver<br>
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[http://hdl.handle.net/1721.1/32535 URL] (open access!)
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Latest revision as of 21:10, 29 June 2017

< Back to Plasmid backbones


   
Plasmid backbones/Assembly of protein fusions
Part assembly System operation Protein expression Assembly of protein fusions Part measurement Screening of part libraries Building BioBrick vectors DNA synthesis Other standards Archive
Or get some help on plasmid backbones.

Protein engineers often need to do in-frame assemblies in order to assemble together different signal sequences, proteins domains, and protein tags. BioBrick™ standard assembly is not well-designed for this task because the scar sequence formed by the SpeI-XbaI ligation is 8 base pairs long, so assembly of protein domains causes frameshifts. As a result, different groups have developed modified or alternative assembly schemes to facilitate assembly of protein domains.

Plasmid backbones that adhere to the Silver standard ([http://bbf.openwetware.org/RFC.html BBF RFC] 23)

SilverFusionScheme.png

Pam Silver's lab has developed Assembly standard 23, often called the Silver standard, for assembling protein domains. It relies on shortening the BioBrick prefix and suffix each by 1 base pair such that the resulting SpeI-XbaI scar is only 6 base pairs long and protein domains can be assembled in frame.

As shown in figure 3 (right), Assembly standard 23 results in the inclusion of a Thr-Arg (ACT AGA) two amino acid junction between protein domains.

One disadvantage of the Assembly standard 23 is that the codon AGA which encodes Arginine is a rare codon in E. coli. Rare codons can prevent over-expression of proteins in E. coli.

See [http://openwetware.org/wiki/The_BioBricks_Foundation:Standards/Technical/Formats The BioBricks Foundation wiki] for a discussion and comparison of different technical standards.

CarolineAjoFranklingPhoto.jpg
 Caroline Ajo-Franklin developed the Silver lab protein fusion vectors BBa_J63009 and BBa_J63010 in Pam Silver's lab.



There are no parts for this table


References

A New BioBrick Assembly Strategy Designed for Facile Protein Engineering
MIT SBWG Technical Reports, 2006 Apr 20
Ira Philips, Pam Silver
[http://hdl.handle.net/1721.1/32535 URL] (open access!)

Plasmid backbones that adhere to the Freiburg standard ([http://bbf.openwetware.org/RFC.html BBF RFC] 25)

The 2007 Freiburg iGEM team proposed an extension of the original BioBrick assembly standard that enables in-frame assembly of protein domains. Two restriction sites are added within the original BioBrick prefix and suffix sites. These additional sites provide compatible ends, can be employed using the same cloning strategy as for the standard restriction sites, and code for amino acids suitable for linkers:

Prefix and suffix

               Prefix                                      Suffix
5' GAATTC GCGGCCGC T TCTAGA TG GCCGGC [part] ACCGGT TAAT ACTAGT A GCGGCCG CTGCAG 3'
   EcoRI    NotI      XbaI Met NgoMIV         AgeI   *    SpeI     NotI    PstI
                              (=NgoMI)

"N-part" prefix

5' GAATTC GCGGCCGC T TCTAG [ATG.part]
   EcoRI    NotI     XbaI

Scar

Assembling two parts based on the new Freiburg standard restriction sites leaves the following scar: 

5' [part A] ACCGGC [part B] 3'
             T  G

See [http://openwetware.org/wiki/The_BioBricks_Foundation:Standards/Technical/Formats The BioBricks Foundation wiki] for a discussion and comparison of different technical standards.

RaikGruenbergPhoto.jpg Raik Gruenberg developed the protein fusion vectors BBa_J18901, BBa_J18902 and BBa_J18903 that adhere to the Freiburg standard.


There are no parts for this table


References

  • [http://hdl.handle.net/1721.1/45140 BBF RFC 25: Fusion Protein (Freiburg) Biobrick assembly standard] by Kristian M. Müller, Katja M. Arndt, the 2007 Freiburg iGEM team, and Raik Grünberg

<biblio>

  1. Nishikubo pmid=16140408
  2. Pfleger pmid=16247774
  3. Varshavsky pmid=8901547

</biblio>

  • [http://www.neb.com/nebecomm/products_Intl/productR0564.asp NgoMIV at NEB]