Difference between revisions of "Part:BBa K3396017"

Latest revision as of 18:59, 27 October 2020

Replaceable1-Replaceable2-P2A-Truncated Trim21-Replaceable3

CMV->Replaceable1->linker->Replaceable2->P2A->Truncated Trim21->Replaceable3->BGH

This part is a composite part providing Golden Gate assembly based rapid construction of PREDATOR Pro plasmids for controlled degradation of specific target protein.

Usage and Biology

Our favorite composite part BBa_K3396012 is a plasmid platform on which different targeting domains and protein dimerization pairs can be easily installed into the Predator Pro system. The subpart BBa_K3396007 functions as the E3 ligase to degrade target protein. The protein region BBa_K3396014 and BBa_K3396015 can be replaced into different protein dimerization pairs in order to achieve signal responsiveness. The BBa_K3396013 part can be replaced by other targeting domains (ScFv. Nanobody, ligand, etc.) to mediate targeted degradation. The parts BBa_K3396013- BBa_K3396015 were designed with flanking BsmBI, BsaI and AlwI sites for the scarless replacement of these parts.

Special Design

As a member of the collection Predator system, special designs were taken for to optimize the applicability and adaptivity of such parts. Specifically, a novel designed substitution system, through which, two proteins could be fused with their corresponding fragment at the same time using Golden-Gate Assembly, was introduced to dramatically simplify the cloning process.

Characterization

We use Golden-Gate Assembly to replace Replaceable-1 with FRB fragment, Replaceable-2 with FKBP-FKBP fragment, Replaceable-3 with GFPnano fragment. This system can be used to degrade the specific GFP. This new part is registered as BBa_K3396010. To test whether the GFP levels can be tuned and continuously regulated by rapamycin, co-transfection of a EGFP expressing plasmid and Predator Pro system were performed. Result showed that there was a significantly decrease in GFP fluorescence (Fig 2A) in BBa_K3396010 expressing group comparing to the control group. Florescence quantification showed that GFP was significantly degraded to about 13% of the original level with the appearance of BBa_K3396010, which confirmed that GFP Predator could be used to degrade target protein with high efficiency (Figure 2B).

Figure 1.(A) Fluorescence images and intensity quantification of BBa_K3396009 transfected group and its negative control group. HEK-293T cells in both groups were transfected with GFP-expression plasmid. (B) Western blotting determining the expression level of GFP in HEK-293T cells transfected with BBa_K3396009.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1106
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1063
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 840
Illegal BsaI site found at 1789
Illegal BsaI.rc site found at 786

@@ Line 7: / Line 7: @@
 This part is a composite part providing Golden Gate assembly based rapid construction of PREDATOR Pro plasmids for controlled degradation of specific target protein.
-<!-- Add more about the biology of this part here
 ===Usage and Biology===
+Our favorite composite part BBa_K3396012 is a plasmid platform on which different targeting domains and protein dimerization pairs can be easily installed into the Predator Pro system. The subpart BBa_K3396007 functions as the E3 ligase to degrade target protein. The protein region BBa_K3396014 and BBa_K3396015 can be replaced into different protein dimerization pairs in order to achieve signal responsiveness. The BBa_K3396013 part can be replaced by other targeting domains (ScFv. Nanobody, ligand, etc.) to mediate targeted degradation. The parts BBa_K3396013- BBa_K3396015 were designed with flanking BsmBI, BsaI and AlwI sites for the scarless replacement of these parts.
+===Special Design===
+As a member of the collection Predator system, special designs were taken for to optimize the applicability and adaptivity of such parts. Specifically, a novel designed substitution system, through which, two proteins could be fused with their corresponding fragment at the same time using Golden-Gate Assembly, was introduced to dramatically simplify the cloning process.
+===Characterization===
+We use Golden-Gate Assembly to replace Replaceable-1 with FRB fragment, Replaceable-2 with FKBP-FKBP fragment, Replaceable-3 with GFPnano fragment. This system can be used to degrade the specific GFP. This new part is registered as BBa_K3396010.
+To test whether the GFP levels can be tuned and continuously regulated by rapamycin, co-transfection of a EGFP expressing plasmid and Predator Pro system were performed. Result showed that there was a significantly decrease in GFP fluorescence (Fig 2A) in BBa_K3396010 expressing group comparing to the control group. Florescence quantification showed that GFP was significantly degraded to about 13% of the original level with the appearance of BBa_K3396010, which confirmed that GFP Predator could be used to degrade target protein with high efficiency (Figure 2B).
+<html>
+<p><img src="https://static.igem.org/mediawiki/parts/e/e8/T--NUDT_CHINA--BBa_K3396017_Fig3.png" alt="" width="500" /></p>
+</html>
+Figure 1.(A) Fluorescence images and intensity quantification of BBa_K3396009 transfected group and its negative control group. HEK-293T cells in both groups were transfected with GFP-expression plasmid. (B) Western blotting determining the expression level of GFP in HEK-293T cells transfected with BBa_K3396009.
+===Sequence and Features===
-<!-- -->
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K3396017 SequenceAndFeatures</partinfo>