Difference between revisions of "Part:BBa K4225012"

 
 
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<partinfo>BBa_K4225012 short</partinfo>
 
<partinfo>BBa_K4225012 short</partinfo>
  
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This is a composite part that is mad by 2 other composite parts, Ptac-TEV (BBa_K4225008) and Pc-RFP-cs-LVA (BBa_K4225012).
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<br>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.
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<br>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
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<br>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
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<br>All constructs were successfully cloned, digestion checked and sequenced. The characterisation of degradation rescue mechanism using fluorescent assay is in progress.
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<center>[[Image:BBa_K4225012_image_1.jpg|500px]]</center> <center>
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<b>Figure 1.</b>  Ptac-TEV-Pc-RFP-CS-LVA at high concentration of TEV </center>
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Please click this link to see the full animation. [https://2022.igem.wiki/hkust/design Design.]
  
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
  
 
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Latest revision as of 13:53, 12 October 2022


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.

BBa K4225012 image 1.jpg
Figure 1. Ptac-TEV-Pc-RFP-CS-LVA at high concentration of TEV

Please click this link to see the full animation. Design.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1020
    Illegal NheI site found at 1043
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 855
    Illegal XhoI site found at 165
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1701
    Illegal AgeI site found at 1813
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 439
    Illegal SapI.rc site found at 787