Difference between revisions of "Part:BBa K4229067"

Revision as of 12:25, 12 October 2022

mTurquoise2 with C-terminal SnoopCatcher 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 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>
-mTurqouise2 is with mVenus2(BBa_K4229066) 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).
+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. You can see a fluorescent signal in the bacteria that expressed either of those two. You can see foci (especially well with the full wiffleball) for both of those reporter genes:
+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:
 [[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]]
@@ Line 65: / Line 64: @@
-It was later confirmed that in fact mTurqouise2 was cached by T1 by a western blot comparing the full wiffleball and the minimal wiffleball.
+It was later confirmed that in fact mTurqouise was cached by T1 by a western blot comparing the full wiffleball and the minimal wiffleball.
 [[File:MTurquoiseWestern.png|800px|thumb|left|: Western Blot of the BMC minimal wiffleball (pT1spysnp) and full wiffleball (pT1spysnpT2T3) + mTurquoise2]]