Part:BBa_K390501

LacPromoter/RBS/PhaP/HlyA/Terminator

This is a composite part that was created to allow us to test the functionality of the HlyA signal peptide. Most significantly, the proof of concept of the phasin/PHA complex secretion was verified using the HlyA targeted phasin. The HlyA 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.

NOTE: The scar between the protein and the signal peptide is not presented correctly in the sequence below. The scar is the Silver-fusion scar (ACTAGA), which allows for in-frame fusion. Additionally, the scars before the signal peptide and after the protein are affected by these parts each having the Silver-fusion prefix and suffix.

Sequence and Features

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]

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.

Cells containing the genes necessary for PHA production, HlyBD membrane proteins, and the HlyA targeted phasin (BBa_K390501) were grown in M9 minimal media. Cells containing the genes for PHA production and the HlyBD membrane proteins were grown as a negative control. After 30 hours of growth, secreted PHA was isolated and analyzed using quantitative 1H NMR. Differences in intensity of the 1H NMR spectra indicate that PHA is released into the extracellular media when HlyA targeted phasin is expressed. We have reason to believe that this platform can be optimized to significantly reduce costs associated with commercial PHA production. This additional work further validates the parts created by USU’s 2009 team and the potential for their implementation in other cellular product recovery.

Fig 1: 1H NMR spectra with PHA characteristic peaks amplified. The top spectrum reports PHA isolated from a culture containing HlyA targeted phasin. The bottom spectrum reports PHA in the negative control that is producing PHA but not secreting phasin. Notice the differences in intensity (y-axis).