Coding
Part:BBa_K4247025:Design
Designed by: Matteo Soana Group: iGEM22_UCopenhagen (2022-09-30)
mCherry-SnoopCatcher
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.