Part:BBa_K4437503

B0034-FLAG-phasin-HlyA (E. coli)

Usage and Biology

Phasin is an intracellular protein native to PHB-producing bacteria, which coats and stabilises polyhydroxybutyrate (PHB) granules in the bacterial cytoplasm, preventing the aggregation of the newly formed polymer [1]. By adding the C-terminus of the HlyA gene to the end of the phasin molecule, the resulting HlyA-tagged phasin molecule -- and the PHB granules to which it is bound -- can be secreted via the single-step Type I secretion system (TISS) in gram-negative bacteria [2]. Building upon the University of Calgary 2017 team’s BBa_K2260002 by substituting the original RBS (BBa_B0034) for a relatively stronger RBS (BBa_B0030), this part is designed to increase phasin-HlyA expression, and the subsequent yield of secreted PHB.

Design

Using the original FLAG-phasin-HlyA part (BBa_K2260002) as a template, the B0034 (BBa_B0034) RBS was substituted with the B0030 (BBa_B0034) RBS. The restriction enzyme cut sites for BstAPI and BstBI were also added, making the part compatible for digestion and ligation into the Tokyo Tech 2012 team’s BBa_K934001 plasmid for PHB expression.

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]

Characterization

We have successfully amplified the B0030-FLAG-phasin-HlyA part and ligated it into the PHB (BBa_K934001) plasmid. These preliminary confirmations were performed with restriction digests and diagnostic gel electrophoresis. Future directions for the charcterization of this part include its transformation into the BL21 E. coli expression vector, and a subsequent SDS-PAGE and Western blot to confirm the production of phasin. These results will be compared to the phasin expression levels of versions of the part outfitted with weaker RBS types. Finally, in-vitro PHB production assays will be used to quantify resulting yields of secreted PHB.

Lane 1 contains a 2log 1000bp ladder. Lanes 2-4 contain the B0030-FLAG-phasin-HlyA insert in the BBa_K934001 plasmid, transformed in and purified from TOP10 E. coli. Lanes 2 and 4 indicate banding at the anticipated range of approximately 7100bp, indicating the successful insertion and transformation of the BBa_K4437503 insert.

References

1. Brown B, Immethun C, Wilkins M, Saha R. Biotechnical applications of phasins: Small proteins with large potential. Renewable and Sustainable Energy Reviews. 2022 Apr 1;158:112129.

2. Thomas S, Holland IB, Schmitt L. The Type 1 secretion pathway — The hemolysin system and beyond. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2014 Aug 1;1843(8):1629–41.