Part:BBa_K342002:Design

Phasin-Phasin Intein (for silver standard fusion)

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Unknown
• 23
  INCOMPATIBLE WITH RFC[23]

  Unknown
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1279
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

This part is a self cleavable fusion protein. It has been designed thanks to the results of the publication Banki et al.

The phasin protein bind 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 cleavable protein which breaks under PH=6 and allows the release od the fused protein at the end of the sequence

The prefix biobrick standard used contains a perfect RBS.
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 in between are the same as the one described in Banki et al.

This part should had been synthesized by Mr gene in once but due to technical issues we had to redesign two separates fusion biobrick and ligate them.

The sequencing of this part confirm the expected sequence.

Source

Phasin protein from Ralstonia eutropha H16
Optimized intein from M. 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