Difference between revisions of "Part:BBa K4229067"

Revision as of 23:35, 13 October 2022

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

This BioBrick was used as a fluorescent reporter fused with a SpyCatcher, enabling recruitment by the 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.

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 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]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4229067 short</partinfo>
-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.
+This BioBrick was used as a fluorescent reporter fused with a SpyCatcher, enabling recruitment by the SpyTag.
-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:
+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.
-[[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]]
+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 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]]