Composite

Part:BBa_K2739010:Design

Designed by: Lok In Lo   Group: iGEM18_Edinburgh_OG   (2018-10-09)
Revision as of 20:33, 16 October 2018 by Owen-lo 923 (Talk | contribs)

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Hybrid promoter-PhaCB-Bktb


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 69
    Illegal NheI site found at 92
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1041
    Illegal BglII site found at 1866
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 347
    Illegal NgoMIV site found at 418
    Illegal NgoMIV site found at 1018
    Illegal NgoMIV site found at 1330
    Illegal NgoMIV site found at 1609
    Illegal NgoMIV site found at 2768
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2836
    Illegal BsaI site found at 3898


Design Notes

This is a composite part, which developed based on the BBa_K2739008 (Btkb) and BBa_K2739011 (Hybrid-promoter-phaCB).

For the Bktb part, its length is 1185bp and excess the 1kb length limit of the IDT sponsor limitation to be generated. Therefore two overlapped fragments of Bktb were then designed and generated by IDT. A set of PCR primer were then design to generate the whole Bktb through the PCR amplification. T--Edinburgh_OG--Notebook_-_bktb_owen1.png

Figure 1. B1 and B2 fragments were codon-optimised for E. coli and synthesised by IDT. The 696 bp overlap was in green and restriction enzyme sites were coloured by red. Two pairs of primers were ordered from IDT to amplify bktB, among which B1 forward primer and B2 reverse primer were used to amplify bktB fragments.

BBa_K2739011 (Hybrid-promoter-phaCB) is created using the BBa_K1149051 (Hybrid promoter-phaCAB) as the template. A set of primer is designed to cut the phaA from the BBa_K934001 (phaC1-A-B1) biobrick and then ligated back.


Source

BBa_K2739008 (Btkb) used in the part were created through IDT. The part of the btkb original sequence would be accessed in NCBI: NC_015726.1. Bktb sequence is codon optimised by IDT.

Hybrid-promoter-phaCB was developed from BBa_K1149051.

References

1. Poehlein,A., Kusian,B., Friedrich,B., Daniel,R. and Bowien,B. (2011) Complete genome sequence of the type strain Cupriavidus necator N-1. J. Bacteriol. 193 (18)