Part:BBa_K4738013
TorA-PhaP-His6 fusion protein with T7 promoter
This year, our project focuses on developing an efficient method for secreting the bioplastic poly(3-hydroxybutyrate), also known as PHB, from E. coli. Our part collection involves 3 basic (BBa_K4738000 to BBa_K4738002) and 14 composite parts (BBa_K4738010 to BBa_K4738016 - Not GFP-tagged and BBa_K4738020 to BBa_K4738026 - GFP-tagged). These parts include both the components of various secretion devices for PHB, and the secretion devices themselves.
These parts enable the ease of assembly for fusion proteins that code for the production and secretion of the bioplastic poly(3-hydroxybutyrate) from E. coli, as well as further exploration into newer secretion methods previously unused for a phasin secretion approach. The secretion tags include the first vesicle nucleating peptides to be added into the Registry (VNp2, VNp6, VNp15, BBa_K4738000 to BBa_K4738002). Furthermore, we provide some insight into the creation of fusion proteins that can secrete the PHB efficiently, and the importance of peptide tag positioning in bacterial and VNp-based secretion.
This part is a fusion protein of a N-terminal TorA secretion tag (BBa_K3114005), Phasin (BBa_K2739004), and a C-terminal polyhistidine tag (BBa_K128005). This part constitutively produces a TorA-PhaP-His6 fusion protein, which secretes through the TorA Type 2 twin-arginine translocation (Tat) bacterial secretion pathway. The His6 tag can be used to purify and qualitatively analyze the secreted and intracellular phasin.
We used this part to verify the secretion of TorA-tagged phasin from E. coli. The TorA (trimethylamine N-oxide reductase A) system is beneficial since it favors folded proteins, which may confer an advantage when secreting larger products such as PHB granules, but has lower efficiency of extraction when compared to methods such as chemical and thermal cell lysis. We transformed this part inside the pSB1A3 plasmid into a culture of BL21 E. coli and allowed it to constitutively produce and secrete phasin over the course of several days. We used this part to verify the estimated yield from secreting purely phasin through the TorA system by purifying the phasin using a nickel spin column and qualitatively analyzing the sample.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 48
Illegal BsaI.rc site found at 25
Illegal BsaI.rc site found at 180
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