Difference between revisions of "Part:BBa K771003"

Revision as of 07:08, 29 September 2012

Membrane Anchor 3 :ssDsbA-PDZ Ligand-LGT-SH3 Ligand

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].

Overview: Membrane Scaffold System

Demonstration of the whole Membrane Scaffold device. Membrane Anchor 2, 3, 4, 5 consitutively aggregate while Membrane Anchor 1 and 6 only aggregate with constitutive cluster (Membrane Anchor 2, 3, 4, 5) when certain signal is present.

Backgroud

Constitutive Aggregation

Membrane Anchor 2 (with and without MS) BBa_K771002,BBa_K771007and Membrane Anchor 3 constitutively aggregate through SH3 domain(BBa_K771107) and SH3 ligand (BBa_K771108).

Membrane Anchor 3 and Membrane Anchor 4 BBa_K771004 constitutively aggregate through PDZ domain(BBa_K771103) and PDZ ligand (BBa_K771104).

Design

Membrane Anchor 3 could constitutively aggregate with Membrane Anchor 2(with and without MS) and 4[BBa_K771002] [BBa_K771004] through interacting domain and ligand.

Characterization

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

The result shows that Membrane Anchor 2 without MS2 and Membrane Anchor 3 could constitutively aggregate through SH3 domain and ligand.

Membrane Anchor 4 and Membrane Anchor 3

The result shows that Membrane Anchor 4 and Membrane Anchor 3 could constitutively aggregate through PDZ domain and ligand.

Related Biobrick

<br\>Membrabe Anchor 1([BBa_K771001]) <br\>Membrabe Anchor 2([BBa_K771002]) <br\>Membrabe Anchor 4([BBa_K771004]) <br\>Membrabe Anchor 5([BBa_K771005]) <br\>Membrabe Anchor 6([BBa_K771006])

Reference

Dueber, J. E., G. C. Wu, et al. (2009). "Synthetic protein scaffolds provide modular control over metabolic flux." Nature biotechnology 27(8): 753-759.

Sequence and Features