Difference between revisions of "Part:BBa K4229067"
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We used this fusion protein to test the functionality of our compartments: the minimal wiffleball (BBa_K4229047), full wiffleball (BBa_K4229049), and SPD-5 (BBa_K4229078). All compartmentalization proteins have an N-terminal SpyTag.  
 
We used this fusion protein to test the functionality of our compartments: the minimal wiffleball (BBa_K4229047), full wiffleball (BBa_K4229049), and SPD-5 (BBa_K4229078). All compartmentalization proteins have an N-terminal SpyTag.  
  
−
A fluorescent signal in the bacteria that expressed either protein could be detected. Upon co-expression with compartmentalization proteins, foci were formed (especially well with the full wiffleball) with both reporter genes:
+
A fluorescent signal in the bacteria that expressed either protein could be detected. Upon co-expression with compartmentalization proteins, foci were formed (especially well with the full wiffleball) with both reporter genes:
 
[[File:MVenusmTurquoise.png|800px|thumb|left|Fluorescent microscopy of T1 catching the mVenus2 and mTurquoise2 when the minimal or full wiffleball construct is expressed: A. Controls for induction; B. T1 with and without the Spy/Snp tags; scale bar = 5µm]]
 
[[File:MVenusmTurquoise.png|800px|thumb|left|Fluorescent microscopy of T1 catching the mVenus2 and mTurquoise2 when the minimal or full wiffleball construct is expressed: A. Controls for induction; B. T1 with and without the Spy/Snp tags; scale bar = 5µm]]
  

Revision as of 23:44, 13 October 2022


mTurquoise2 with C-terminal SnoopCatcher regulated by tetA/B promotor

This composite BioBrick was used to visualize protein targeting into microcompartments by fluorescent microscopy. The SnoopCatcher is fused to the C-term of mTurquoise2.

We used this fusion protein to test the functionality of our compartments: the minimal wiffleball (BBa_K4229047), full wiffleball (BBa_K4229049), and SPD-5 (BBa_K4229078). All compartmentalization proteins have an N-terminal SpyTag.

A fluorescent signal in the bacteria that expressed either protein could be detected. Upon co-expression with compartmentalization proteins, foci were formed (especially well with the full wiffleball) with both reporter genes:

Fluorescent microscopy of T1 catching the mVenus2 and mTurquoise2 when the minimal or full wiffleball construct is expressed: A. Controls for induction; B. T1 with and without the Spy/Snp tags; scale bar = 5µm





























It was later confirmed that in fact mTurqouise was cached by T1 by a western blot comparing the full wiffleball and the minimal wiffleball.

: Western Blot of the BMC minimal wiffleball (pT1spysnp) and full wiffleball (pT1spysnpT2T3) + mTurquoise2















With that, we can say that our mTurquiose2 with the snoopCatcher is successfully expressed and caught by our compartment.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 59
    Illegal PstI site found at 856
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 59
    Illegal PstI site found at 856
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 59
    Illegal BglII site found at 68
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 59
    Illegal PstI site found at 856
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 59
    Illegal PstI site found at 856
    Illegal AgeI site found at 121
    Illegal AgeI site found at 1017
  • 1000
    COMPATIBLE WITH RFC[1000]