Difference between revisions of "Part:BBa K342002:Design"
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Phasin protein from <i>Ralstonia eutropha H16</i><br> | Phasin protein from <i>Ralstonia eutropha H16</i><br> | ||
− | Optimized intein from <i>Mycobacterium tuberculosis</i> | + | Optimized intein from <i>Mycobacterium tuberculosis (Mtu)</i> recA<br> |
Ligation of two synthetized parts : Phasin and Phasin-Intein | Ligation of two synthetized parts : Phasin and Phasin-Intein | ||
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Revision as of 17:37, 7 November 2010
Phasin-Phasin Intein (for silver standard fusion)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1693
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1279
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
This part is a self-cleaving fusion protein. It has been designed thanks to the results of the publication
Banki et al.
The phasin protein binds to the granule membrane. The second phasin makes the binding stronger and then increases the rate of purified protein during the step of granules extraction. The intein is a self-cleaving protein which breaks under pH=6 and allows the release of the protein of interest fused at the end of the sequence The prefix biobrick standard used contains a perfect RBS. This part should have been synthesized by Mr gene in once but due to technical issues we had to redesign two separated fusion biobricks and ligate them. The sequencing of this part confirmed the expected sequence.
The suffix is a silver standard fusion suffix: 5' ACTAGT A GCGGCCG CTGCAG 3'.
The phasin sequence is the same as the biobrick BBa_K208001 from the Utah 2009 team.
The intein is from the self-cleaving protein sequence delta.I-CM mini intein from mycobacterium tuberculosis described in this patent.
The intergenic regions are the same as the one described in Banki et al.
Source
Phasin protein from Ralstonia eutropha H16
Optimized intein from Mycobacterium tuberculosis (Mtu) recA
Ligation of two synthetized parts : Phasin and Phasin-Intein
References
"Novel and economical purification of recombinant proteins: Intein-mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association", Mahmoud Reza Banki, Tillman U.Gerngross and David W. Wood, 2005, Protein Science, 14:1387–1395