Coding

Part:BBa_K2022012:Design

Designed by: Jessica Mazalo, Daniel Winter   Group: iGEM16_UNSW_Australia   (2016-10-12)


INPNC-GFP, i.e. membrane-anchored GFP


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 952
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 72
    Illegal NgoMIV site found at 405
    Illegal AgeI site found at 823
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1604


Design Notes

Pennsylvania (2012) designed their INPNC part with the BamHI site, to make it readily fusible to other proteins; we thank them for their contribution in making the assembly and design processes as simple as possible.

As such, the only design consideration was to use the oligonucleotide 5'-aggcggatccggtatgcgtaaaggagaagaacttttcactg-3' to add a BamHI site upstream of the GFP open reading frame. Here, the first 5' 10nt include the 4nt followed by the 6nt recognition sequence of BamHI, with the former included as BamHI doesn't cut efficiently at the end of strands. We also included an extra codon (ggt) between the end of INPNC and the start of GFP.


Source

To make this part, we combined BBa_K811005 (INPNC) with BBa_E0040 (GFP).

First, we digested BBa_K811005 with BamHI and PstI.

Then, we PCR amplified BBa_E0040 using Suffix-R2, and a forward oligonucleotides that replaced the prefix with a BamHI site (aggcggatccggtatgcgtaaaggagaagaacttttcactg). This PCR product was then also digested with BamHI and PstI.

These two products were then ligated together, and transformed into E. coli host (DH5-alpha derivative)

References