Difference between revisions of "Part:BBa K4229066"
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<partinfo>BBa_K4229066 short</partinfo>
 
<partinfo>BBa_K4229066 short</partinfo>
  
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mVenus2 is with mTurqouise2(BBa_K4229067) one of our reporter genes, with which we tested the building and catching of the minimal(BBa_K4229047) and full wiffleball(BBa_K4229049) and SPD5 (BBa_K4229078).
 
  
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The snoop/spyTag snoop/spyCatcher was tested with those two reporter genes. You can see an flourecent signal in the bacteria which expressed wither of those two:
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mVenus2 (together with mTurquoise2,[BBa_K4229064]) was used as a reporter by fusing it with a SpyCatcher, enabling recruitment by the SpyTag. We used this fusion protein to test the functionality of our compartments: the minimal (BBa_K4229047) and full wiffleball (BBa_K4229049), and SPD5 (BBa_K4229078). All compartmentalisation proteins have an N-terminal SpyTag. For experimental data please refer to the data listed in Biobrick BBa_K4229067.
  
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[[File:MVenusmTurquoise.png|800px|thumb|left|Fluorescent microscopy of T1 catching the mVenus2 and mturquoise2 when the minimal or full BMC construct is expressed: A Controls for induction; B T1 with and without the Spy/Snp tags; scalebar 5µm]]
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The Snoop/SpyTag Snoop/SpyCatcher was tested with those two reporter genes. A fluorescent signal in the bacteria that expressed either protein could be detected.Upon co-expression with compartmentalisation proteins, foci were formed (especially well with the full wiffleball) with both reporter genes:
  
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[[File:MVenusmTurquoise.png|800px|thumb|left|Fluorescent microscopy of T1 catching the mVenus2 and mTurquoise2 when the minimal or full BMC construct is expressed: A Controls for induction; B T1 with and without the Spy/Snp tags; scalebar 5µm]]
  
  
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It was later confirmed that in fact mVenus was cached by T1 by a western blot comparing the full wiffleball with and without the necessary tags.
  
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[[File:FullT1Western.png|800px|thumb|left|Western Blot comparison of the BMC minimal wiffleball with and w/o tags (pHT1) + mVenus2]]
  
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With that, we can say that our mVenus tagged with the SpyCatcher is successfully expressed and caught by our compartment.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 12:23, 12 October 2022


mVenus with N-terminal spyCatcher regulated by tetA/B promotor


mVenus2 (together with mTurquoise2,[BBa_K4229064]) was used as a reporter by fusing it with a SpyCatcher, enabling recruitment by the SpyTag. We used this fusion protein to test the functionality of our compartments: the minimal (BBa_K4229047) and full wiffleball (BBa_K4229049), and SPD5 (BBa_K4229078). All compartmentalisation proteins have an N-terminal SpyTag. For experimental data please refer to the data listed in Biobrick BBa_K4229067.

The Snoop/SpyTag Snoop/SpyCatcher was tested with those two reporter genes. A fluorescent signal in the bacteria that expressed either protein could be detected.Upon co-expression with compartmentalisation 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 BMC construct is expressed: A Controls for induction; B T1 with and without the Spy/Snp tags; scalebar 5µm





























It was later confirmed that in fact mVenus was cached by T1 by a western blot comparing the full wiffleball with and without the necessary tags.

Western Blot comparison of the BMC minimal wiffleball with and w/o tags (pHT1) + mVenus2















With that, we can say that our mVenus tagged with the SpyCatcher is successfully expressed and caught by our compartment.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 59
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 59
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 59
    Illegal BglII site found at 68
    Illegal XhoI site found at 1075
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 59
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 59
    Illegal AgeI site found at 508
  • 1000
    COMPATIBLE WITH RFC[1000]