Difference between revisions of "Part:BBa K771402"

Latest revision as of 16:22, 2 October 2012

split EGFP 1 with Membrane Anchor 2 (without MS2)

split EGFP 1 with Membrane Anchor 2 (without MS2)

Membrane anchor 2 (without MS2) consists of SH3 domain(BBa_K771107) and membrane protein Lgt(BBa_K771102). To testify the constitutive aggregation of Membrane Anchor 2 without MS2 and Membrane Anchor 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 split EGFP1 (BBa_K771113) and split EGFP 2(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.

Sequence and Features