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™ 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™ 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== | ==Plasmid backbones that adhere to the Silver standard== | ||
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===References=== | ===References=== | ||
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==Plasmid backbones that adhere to the Freiburg standard== | ==Plasmid backbones 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! | Unfortunately, the Registry does not currently have any plasmid backbones that comply with the Freiburg standard. If you have some, please contribute! | ||
Revision as of 03:35, 2 February 2009
| 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
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.
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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!)
Plasmid backbones 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!