Part:BBa_K4437502

B0030-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, this part was produced by substituting the original RBS (BBa_B0034) for the B0030 (BBa_B0030) RBS, which is relatively stronger. As such, this part can be used as a test to assess if increasing the strength of the RBS upstream of the FLAG-phasin-HlyA sequence, and the resulting expression levels of this protein, subsequently increases the 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_B0030) 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 assays. The FLAG protein allows for facilitated purification, and for characterization assays such as the Western blot.

File:02-constructmap.png

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Figure 1. An overview of the BBa_K4437501 construct map.

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

<p> We have successfully amplified the B0030-FLAG-phasin-HlyA part, ligated it into the PHB (BBa_K934001) plasmid, and transformed and expressed this plasmid in TOP10 E. coli cells. These preliminary confirmations were performed with restriction digests and diagnostic gel electrophoresis. Future directions for the characterization of this part include the transformed of the recombinant DNA into BL21 (DE3) E. coli cells, the purification of this product, and the characterization of the desired FLAG-phasin-HlyA protein product on an SDS-PAGE using anti-FLAG antibodies. Additionally, this includes DNA sequencing, and the use of in-vitro PHB production assays to quantify resulting yields of secreted PHB.

Figure 2. Agarose electrophoresis of purified DNA. 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_K4437502 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.