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 |
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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™ 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
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.
As shown in figure 3 (right), the Silver standard results in the inclusion of a Thr-Arg (ACT AGA) two amino acid junction between protein domains.
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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Caroline Ajo-Franklin developed the Silver lab protein fusion vectors BBa_J63009 and BBa_J63010 in Pam Silver's lab. |
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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!)
Plasmids that adhere to the Freiburg standard
Unfortunately, the Registry does not currently have any plasmid backbones that comply with the Freiburg standard. If you have some, please contribute!