Coding

Part:BBa_K4247025:Design

Designed by: Matteo Soana   Group: iGEM22_UCopenhagen   (2022-09-30)


mCherry-SnoopCatcher


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
    COMPATIBLE WITH RFC[1000]


Design Considerations

We used PCR to extract mCherry from an existing plasmid and used three parallel PCR reactions with six User primers in total to:

1) Amplify the backbone of the plasmid containing mCherry (pBad)

2) Switch the 6-His tag from C-terminus to N-terminus while amplifying the mCherry fragment, based on our experience that E.coli expresses better proteins with an N-terminal 6-His tag

3) Amplify SnoopCatcher from Mfp151_SnoopCatcher (BBa_K4247021), including a short linker (GGGSG)

We then unified these components via a User reaction.

Hence, mCherry-SnoopCatcher was contained in a pBAD plasmid. The protein was expressed under a promoter that could be induced with Arabinose. We used 0.2% arabinose to induce protein expression.

MCherrySnoopCatcher.png