Difference between revisions of "Part:BBa K771403"

Latest revision as of 16:24, 2 October 2012

split EGFP2 with Membrane Anchor 3

Membrane anchor 3, which consists of interacting SH3 ligand, PDZ ligand and membrane protein Lgt. Membrane Anchor 3 is proved to constitutively aggregate with Membrane Anchor 2 (with and without MS2)and Membrane Anchor 4[BBa_K771002] [BBa_K771004].

To testify the constitutive aggregation of Membrane Anchor 2, 3 and 4, we conducted Fluorescence Complementation Assay.

In fluorescence complementation assay, proteins that are postulated to interact are fused to unfolded complementary fragments of EGFP and expressed in E.coli. Interaction of these proteins will bring the fluorescent fragments within proximity, allowing the reporter protein to reform in its native three-dimensional structure and emit its fluorescent signal. EGFP was split into two halves, named 1EGFP(BBa_K771113) and 2EGFP(BBa_K771114) respectively. If there is interaction between two proteins which were fused with 1EGFP and 2EGFP, it is expected that fluorescence should be observed. Otherwise, no fluorescence could be observed.

Membrane Anchor 2 without MS2 and Membrane Anchor 3

We fused split EGFP 1 and 2 to Membrane Anchor 2 without MS2 and Membrane Anchor 3 to test whether Membrane Anchor 2 without MS2 and Membrane Anchor 3 could constitutively aggregate. Proteins in control group are not expected to aggregate like in experimental group.We coexpressed proteins in experimental group and control group respectively in E.coli. After induction for 6 hours, bacteria samples were taken for fluorescence observation. The result shows that Membrane Anchor 2 without MS2 and Membrane Anchor 3 could constitutively aggregate through SH3 domain and ligand.

Membrane Anchor 3 and Membrane Anchor 4

|We fused split EGFP 1 and 2 to Membrane Anchor 4 and Membrane Anchor 3 to test whether Membrane Anchor 4 and Membrane Anchor 3 could constitutively aggregate. Proteins in control group are not expected to aggregate like in experimental group.We coexpressed proteins in experimental group and control group respectively in E.coli. After induction for 6 hours, bacteria samples were taken for fluorescence observation.The result shows that Membrane Anchor 4 and Membrane Anchor 3 could constitutively aggregate through PDZ domain and ligand.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 905
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 526

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 __NOTOC__
 <partinfo>BBa_K771403 short</partinfo>
-M2-EGFP2
+Membrane anchor 3, which consists of interacting SH3 ligand, PDZ ligand and membrane protein Lgt. Membrane Anchor 3 is proved to constitutively aggregate with Membrane Anchor 2 (with and without MS2)and Membrane Anchor 4[[https://parts.igem.org/wiki/index.php?title=Part:BBa_K771002 BBa_K771002]] [[https://parts.igem.org/wiki/index.php?title=Part:BBa_K771004 BBa_K771004]].
+To testify the constitutive aggregation of Membrane Anchor 2, 3 and 4, we conducted Fluorescence Complementation Assay.
+In fluorescence complementation assay, proteins that are postulated to interact are fused to unfolded complementary fragments of EGFP and expressed in ''E.coli''. Interaction of these proteins will bring the fluorescent fragments within proximity, allowing the reporter protein to reform in its native three-dimensional structure and emit its fluorescent signal. EGFP was split into two halves, named 1EGFP([https://parts.igem.org/wiki/index.php?title=Part:BBa_K771113 BBa_K771113]) and 2EGFP([https://parts.igem.org/wiki/index.php?title=Part:BBa_K771114 BBa_K771114]) respectively. If there is interaction between two proteins which were fused with 1EGFP and 2EGFP, it is expected that fluorescence should be observed. Otherwise, no fluorescence could be observed.
+===Membrane Anchor 2 without MS2 and Membrane Anchor 3===
+[[Image:12SJTU-MA23.png|center|500px]]
+[[Image:12SJTU Anchor GFP12.jpg|450px|center]]
+We fused split EGFP 1 and 2 to Membrane Anchor 2 without MS2 and Membrane Anchor 3 to test whether Membrane Anchor 2 without MS2 and Membrane Anchor 3 could constitutively aggregate. Proteins in control group are not expected to aggregate like in experimental group.We coexpressed proteins in experimental group and control group respectively in ''E.coli''. After induction for 6 hours, bacteria samples were taken for fluorescence observation. The result shows that Membrane Anchor 2 without MS2 and Membrane Anchor 3 could constitutively aggregate through SH3 domain and ligand.
+===Membrane Anchor 3 and Membrane Anchor 4===
+|We fused split EGFP 1 and 2 to Membrane Anchor 4 and Membrane Anchor 3 to test whether Membrane Anchor 4 and Membrane Anchor 3 could constitutively aggregate. Proteins in control group are not expected to aggregate like in experimental group.We coexpressed proteins in experimental group and control group respectively in ''E.coli''. After induction for 6 hours, bacteria samples were taken for fluorescence observation.The result shows that Membrane Anchor 4 and Membrane Anchor 3 could constitutively aggregate through PDZ domain and ligand.
+[[Image:12SJTU_splitegfp34.png|400px|center]]
+[[Image:12SJTU Anchor GFP.jpg|450px|center]]
 <!-- Add more about the biology of this part here