Difference between revisions of "Plasmid backbones/Assembly of protein fusions"

Revision as of 20:57, 6 September 2008


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.

Plasmids 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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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

To be added.

@@ Line 13: / Line 13: @@
 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 the figure 3 (right), the Silver standard results in the inclusion of a Thr-Arg (ACT AGA) two amino acid junction between protein domains.
+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.