Difference between revisions of "Part:BBa K1467200"
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The colonies containing the RFP 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 the flipper reaction is successful the colonies become white as the RFP sequence is replaced by the new part. No part of the BsaI recognition sequence will remain between the BioBrick suffix and the new part. | The colonies containing the RFP 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 the flipper reaction is successful the colonies become white as the RFP sequence is replaced by the new part. No part of the BsaI recognition sequence will remain between the BioBrick suffix and the new part. | ||
| + | iGEM14_NRP-UEA-Norwich have used this part to convert GoldenGate "CDS" (coding sequence) 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. | ||
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| + | [[File:CDS-flipper characterisation.jpg|600px]] | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
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Revision as of 10:12, 6 October 2014
RFP coding device - Golden Gate Module Flipper
This part is a "GoldenGate Flipper" that can be used to convert CDS1 (coding sequence) modules from the GoldenGate MoClo Assembly Standard into standard BioBricks. It consists of the RFP reporter (from 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 CDS1 parts, which begin AATG and end GCTT.
The colonies containing the RFP 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 the flipper reaction is successful the colonies become white as the RFP sequence is replaced by the new part. No part of the BsaI recognition sequence will remain between the BioBrick suffix and the new part.
iGEM14_NRP-UEA-Norwich have used this part to convert GoldenGate "CDS" (coding sequence) 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 793
Illegal AgeI site found at 905 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1082
Illegal BsaI.rc site found at 6