Composite

Part:BBa_K2739006:Design

Designed by: Lok In Lo   Group: iGEM18_Edinburgh_OG   (2018-10-08)
Revision as of 18:21, 17 October 2018 by Owen-lo 923 (Talk | contribs)


proR-PhaR-proP-PhaP-HlyA


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 858
    Illegal SapI.rc site found at 253


Design Notes

This composite part is consisted PhaR and its native promoter + Phasin fused with HlyA signal peptide and its native promoter. This part was further developed based on the BBa K2739000 (proR-PhaR). It was known from previous team that Phasin fused with HlyA signal peptide would result in PHA secretion. Therefore, we would develop the current composite part to investigate if the secretion function of Phasin-HlyA is compatible with phaR and if the improvement of phaR of PHA production is remained in this parts composition.

Sequence of Phasin-HlyA was obtained from previous item team, BBa_K390501(LacP-RBS-PhaP-HlyA-Terminator). Our team found that the end of Phasin's sequence contain a stop codon. The stop codon was then removed and the related part was used to construct the current composite part.

T--Edinburgh_OG_BBa_K2739007--image_5.jpeg Figure 1. The sequence alignment of BBa_K390501, LacP-Phasin-Hly-VR-170918-08-27(The sequencing result of BBa K2739007) and original-phasinHlyA-VR-170918-08-28 (The sequencing result of BBa_K390501) via Benchling. The selected (blue) part of the sequence indicated the stop codon.


Sequence of Phasin's native promoter was isolated from Ralstonia eutropha and not found on iGEM registry. Therefore, this promoter was genereated by IDT.

Sequence of PhaR and its native promoter could be found in iGEM registry, as two separate basic parts (BBa_K108015 and BBa_K108014) and being not available. The source of these sequences were recorded as 1. Regulation of phasin expression and polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha H16. Microbiology. 2002, 148:2413–2426. and 2. NCBI (with no further information), having no mentioned if the sequence was codon optimised.

We access the online NCBI data and found out the current available phaR sequence is being 4 bp different to BBa_K108015; and the functional promoter is being 134bp shorter. Therefore we have used the new phaR and promoter sequence as our new template. Due to the excessive GC in the sequence, we have it codon optimised to E.coli and generated the part through the IDT company service.


T--Edinburgh_OG_BBa_K2739000--image_9.jpeg

Figure 2. Comparison of phaR sequence from iGEM08_Tsinghua and online NCBI. Capital letters: NCBI and Small letter: iGEM08_Tsinghua.

T--Edinburgh_OG_BBa_K2739000--image_10.jpeg

Figure 3. Comparison of phaR sequence before and after codon optimisation. Capital letters: NCBI and Small letter: codon optimised version.


Source

BBa_K108015, BBa_K108014 and NCBI (AM260479.1)

ProR and PhaR Sequence selection based on Reference 1 information.

PhaR sequence codon optimised through IDT. (And Phasin sequence optimised by previous team)

References

1. Yamada M, Takahashi S, Okahata Y, Doi Y, Numata K. (2013 ) Monitoring and kinetic analysis of the molecular interactions by which a repressor protein, PhaR, binds to target DNAs and poly[(R)-3-hydroxybutyrate]. AMB Express. 3(1):6.