Part:BBa_K2260002
Phasin-HlyA
Phasin is an intracellular protein native to PHB-producing bacteria, and binds to PHB deposits within the cell through electrostatic interaction. HlyA is a toxin produced by E. coli that is secreted via an endogenous, single-step type one secretion system. By adding the C-terminus of the HlyA gene to the end of the phasin molecule, the phasin is marked for secretion through the pathway designed for the HlyA. As a result, the phasin molecule binds to intracellular PHB granules and then is secreted due to the HlyA tag, effectively transporting PHB outside of the cell.
This part has been catered towards usage in E. coli, specifically in the strain BL21 DE3, as this strain has endogenous T7 polymerase to complement the T7 promoter we have for our gene. Thus, this sequence has been codon optimized to optimize success with E. coli. Furthermore, restriction sites have been altered in order to make this gene compatible with all RFC assembly standards, ensuring ease of access for future teams.
When designing our part, we used the part BBa_K2018024, designed by the SDU-Denmark 2016 team, as a template. It can be found at https://parts.igem.org/Part:BBa_K2018024.
Our assays showed that our part resulted in a 114% increase of secreted PHB found in the media, compared to PHB found in the media of cells without out part.
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]
| None |