Coding

Part:BBa_K208001:Design

Designed by: Elisabeth Linton   Group: iGEM09_Utah_State   (2009-10-08)

Phasin (PhaP)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Silver-fusion compatible in plasmid pSB3K3. A PstI site was mutated out of the original sequence. Instead of CTGCAG, the site is CTTCAG. This mutation does not affect the amino acid sequence.

The Silver-fusion compatibility means that the prefix and suffix are slightly modified from the original BioBrick prefix and suffix. Specifically, a single base pair is removed from the prefix and suffix, directly next to the part.
The Silver prefix is: 5' GAATTC GCGGCCGC T TCTAGA 3'
The Silver suffix is: 5' ACTAGT A GCGGCCG CTGCAG 3'

The scar that is formed from ligating XbaI and SpeI between two fusion compatible parts is 5' ACTAGA 3', which is six base pairs long and does not contain a stop codon. Therefore, fusion of two proteins can occur by this method. Additionally, this part is still useful and functional for non-fusion applications. There are more design considerations for when using Silver BioBrick parts. See this description by the Silver lab for more information about Silver fusion: http://www.openwetware.org/wiki/Silver:_BB_Strategy

Source

This sequence was isolated from Ralstonia eutropha using PCR and designed primers. The primers were designed so that the Silver-fusion prefix and suffix were overhanging. The final PCR product was then cut and inserted into a BioBrick vector, pSB3K3.

References

1. Maehara A, Ueda S, Nakano H, Yamane T (1999) Analyses of a polyhydroxyalkanoic acid granule-associated 16-kilodalton protein and its putative regulator in the pha locus of Paracoccus denitrificans. J Bacteriol 191:2914-2921
2. York GM, Junker BH, Stubbe J, Sinskey AJ (2001) Accumulation of the PhaP Phasin of Ralstonia eutropha is dependent on production of polyhydroxybutyrate in cells. J Bacteriol 183:4217-4226
2. York GM, Stubbe J, Sinkskey AJ (2002) New insight into the role of the PhaP phasin of Ralstonia eutropha in promoting synthesis of polyhydroxybutyrate. J Bacteriol 183:2394-2397