Difference between revisions of "Part:BBa K4229067"

Latest revision as of 23:56, 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.

Upon co-expression with compartmentalization proteins, foci were formed (especially well with the full wiffleball).

Fluorescent microscopy of T1 catching the mVenus2 and mTurquoise2 when the minimal or full wiffleball construct is expressed; scale bar = 5µm

When testing SnpTag/Catcher system, we found fluorescent foci, visible under the fluorescence microscope. Therefore, we assume that we can see the encapsulation of mTurquoise2 in the BMCs and the formation of the compartment. To confirm these results, further experimental evaluation will be needed.

The covalent catching of mTurquoise2 by the T1 is proven by western blots (Figure 1.7). Surprisingly, the full wiffleball showed insoluble T1 and mTurquoise2-bound T1, although only for some induction conditions. The expression of the minimal and full wiffleball does not seem to influence the formation of insoluble aggerates of the proteins and seems to happen in a more random fashion.

We could show that mTurquoise2 is targeted to microcompartments when coexpressed with full or wiffleball.

:Western Blot showing the formation of the peptide bond between the T1 protein and mTurquoise2 in cells expressing the full and the minimal wiffleball with and w/o tags. The detection of the T1 protein was performed with an anti-his antibody.

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 8: / Line 8: @@
 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:
+Upon co-expression with compartmentalization proteins, foci were formed (especially well with the full wiffleball).
 [[File:MVenusmTurquoise.png|800px|thumb|left|Fluorescent microscopy of T1 catching the mVenus2 and mTurquoise2 when the minimal or full wiffleball construct is expressed; scale bar = 5µm]]
@@ Line 66: / Line 66: @@
+When testing SnpTag/Catcher system, we found fluorescent foci, visible under the fluorescence microscope. Therefore, we assume that we can see the encapsulation of mTurquoise2 in the BMCs and the formation of the compartment. To confirm these results, further experimental evaluation will be needed.
-It was later confirmed that in fact mTurqouise was cached by T1 by a western blot comparing the full wiffleball and the minimal wiffleball.
+The covalent catching of mTurquoise2 by the T1 is proven by western blots (Figure 1.7). Surprisingly, the full wiffleball showed insoluble T1 and mTurquoise2-bound T1, although only for some induction conditions. The expression of the minimal and full wiffleball does not seem to influence the formation of insoluble aggerates of the proteins and seems to happen in a more random fashion.
-[[File:MTurquoiseWestern.png|800px|thumb|left|: Western Blot of the BMC minimal wiffleball (pT1spysnp) and full wiffleball (pT1spysnpT2T3) + mTurquoise2]]
+We could show that mTurquoise2 is targeted to microcompartments when coexpressed with full or wiffleball.
+[[File:MTurquoiseWestern.png|800px|thumb|left|:Western Blot showing the formation of the peptide bond between the T1 protein and mTurquoise2 in cells expressing the full and the minimal wiffleball with and w/o tags. The detection of the T1 protein was performed with an anti-his antibody.]]