Part:BBa_K208038
Lac Promoter/RBS/GeneIII/PhaP/Terminator
This is a composite part that was created to demonstrate phasin secretion. The GeneIII signal peptide was fused to the phasin sequence. Both of these parts are Silver-fusion compatible, as discussed on their individual part pages. The lac promoter was used.
Usage and Biology
PHA-associated proteins, called phasins, strongly interact with the PHA granule surface (York, 2001; Maehara, 1999). Accordingly, PHA recovery may be possible by tagging the phasin protein for translocation. Specifically, the Silver fusion Biobrick standard can be used to create constructs in which a targeting signal peptide sequence is genetically fused to the phasin protein (Phillips, 2006). Fusing a signal peptide to a protein promotes export of the complex out of the cytoplasm (Choi, 2004; Mergulhão, 2005). The interaction of phasin with PHA is required for secretion-based granule recovery because PHA is a non-proteinaceous compound produced by the action of three enzymes (Suriyanmongkol 2007; Verlinden 2007). Consequently, the signal peptide cannot be directly attached PHA granules. The phasin protein with attached signal peptide binds to PHA granules, thereby creating a PHA-phasin-signal peptide complex that may be recognized by the cell for export.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
This figure of an SDS gel shows potential phasin secretion. The geneIII secretion signal sequence fused to the phasin protein was expressed in E. coli cells. The E. coli cells were grown overnight in LB growth media and centrifuged to pellet the cells. Supernatants (5ml) were then concentrated using a Centricon Centriplus concentrator (Amicon, Beverly MA). This process concentrated proteins that were larger than 10kDa and removed molecules smaller than 10kDa. Approximately 20ug of protein were then applied to a SDS polyacrylamide gel to separate the proteins according to size. The gel was then stained with coomassie blue for protein detection. Following SDS polyacylamide gel electrophoresis (PAGE) and subsequent coomassie blue staining of the separated proteins, a protein with an approximate size of 22kDA is observed in the sample from the phasin-expressing E. coli cells that is not present in the control E. coli sample. The phasin protein has been reported by others to migrate on SDS PAGE from 14-28kDa (Pötter, 2002; York, 2002). These results indicate that the GeneIII::phasin expression construct is being produced by the E. coli cells and is being secreted outside the cell into the media.
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