Part:BBa_K4225012
Ptac-TEV-Pc-RFP-CS-LVA
This is a composite part that is made by 2 other composite parts, Ptac-TEV (BBa_K4225008) and Pc-RFP-CS-LVA (BBa_K4225010). It is part of a degradation rescue system to amplify RFP fluorescent output.
RFP is tagged with a potyvirus SSRA C-terminal degradation tag LVA, which can be recognised and degraded by proteases ClpXP and ClpAP, which are endogenous in e coli cytoplasm. As a TEV cut site is placed in between RFP and the LVA tag, when TEV expression is inducted by IPTG, the LVA tag can be cleaved off, rescuing the RFP from degradation
The Degradation rescue system is an effective tool for amplifying the dynamic range of an output. As TEV Proteases have catalytic activity, a single molecule of protease can act upon many molecules of substrate as opposed to a transcription factor that can bind to only one site at a time
All constructs were successfully cloned, digestion checked and sequenced. The characterisation of degradation rescue mechanism using fluorescent assay is in progress.
Please click this link to see the full animation. Design.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1020
Illegal NheI site found at 1043 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 855
Illegal XhoI site found at 165 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1701
Illegal AgeI site found at 1813 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 439
Illegal SapI.rc site found at 787
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