Part:BBa_K1467100

RFP coding device - Golden Gate Module Flipper

This part is a "GoldenGate Flipper" that can be used to convert Pro + 5U (promoter and 5' untranslated leader) modules from the GoldenGate MoClo Assembly Standard into standard BioBricks. It consists of the RFP reporter (from https://parts.igem.org/Part:BBa_J04450 (BBa_J04450) flanked by an inverted pair of BsaI recognition sequences. GoldenGate MoClo parts can be flipped into BrioBrick parts in a one-pot, one-step, digestion-ligation GoldenGate cloning reaction. The image below shows the sequences inserted (in orange) between the RFP and the BioBrick prefix and suffix that enable the flipper to accept MoClo Po + 5U parts, which begin GGAG and end AATG.

The colonies containing this part are clearly red in color under natural light after about 18 hours. Smaller colonies are visibly red under UV. The RFP part does not contain a degradation tag and the RBS is strong.When cloning is successful the colonies become white as the RFP sequence is replaced by the new part. After the reaction is complete, no part of the BsaI recognition sequence will remain between the BioBrick suffix and the part.

iGEM14_NRP-UEA-Norwich have used this part to convert GoldenGate "Pro + 5U" (promoter and 5' untranslated leader) modules from the GoldenGate MoClo Assembly Standard into standard BioBricks. The cloning reaction was a Golden Gate one-step, one-pot reaction where the restriction enzyme, ligase and (intact, uncut) donating and recipient plasmids were all incubated together in a single reaction before transformation into competent E.coli cells. Cloning was very successful as the vast majority of the colonies are white as the RFP sequence was replaced by the new part.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 793
  Illegal AgeI site found at 905
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1082
  Illegal BsaI.rc site found at 6