Difference between revisions of "Part:BBa K771008"

Revision as of 10:57, 2 October 2012

Membrane Anchor 5 without VVD:ssDsbA-LGT-GBD Ligand

Membrane anchor 5 without VVD, which consists of GBD ligand(BBa_K771106) and membrane

protein Lgt(BBa_K771102). Membrane Anchor 5 (with and without VVD) is proved to

constitutively dimerize with Membrane Anchor 4 [BBa_K771004].

Overview: Membrane Scaffold System (without signal induction)

Demonstration of the Membrane Scaffold device (without signal induction). Membrane Anchor 2 (without MS2), 3, 4, 5(without VVD) consitutively aggregate

together.

Backgroud

Constitutive Aggregation

Membrane Anchor 4 (BBa_K771003)and Membrane Anchor 5 (with and without VVD) constitutively

aggregate through GBD domain(BBa_K771105) and GBD ligand

(BBa_K771106).

Characterization

We proved the constitutive interaction between membrane anchor 4 and 5 by fusing split EGFP to membrane anchor 4 and membrane anchor 5 without VVD.

Membrane Anchor 4 and Membrane Anchor 5 without VVD

We fused split EGFP 1 and 2 to Membrane Anchor 4 and Membrane Anchor 5 without VVD to test whether Membrane Anchor 4 and Membrane Anchor 5 without VVD 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 observationThe result shows that Membrane Anchor 4 and Membrane Anchor 5 without VVD could constitutively aggregate through GBD domain and ligand.

Design

Membrane Anchor 5 could constitutively aggregate with Membrane Anchor 4.

Application

We recruired fatty acid biosynthetic pathway involving TesA(BBa_K771301), FabG(BBa_K771302)) , FabI(BBa_K771303), FabZ(BBa_K771304) to put our membrane scaffold system into practice. Fatty Acid productivity is enhanced by 24 fold.

We constructed TesA with Membrane Anchor 2 (without MS2) (BBa_K771305),FabG with Membrane

Anchor 3,(BBa_K771306), FabZ with Membrane Anchor 4(BBa_K771307), FabI with Membrane Anchor 4(without VVD ) (BBa_K771308). Click into each part for more infomation.

Related Biobrick

<br\>Membrabe Anchor 2 without MS2([BBa_K771007]) <br\>Membrabe Anchor 3([BBa_K771003]) <br\>Membrabe Anchor 4([BBa_K771004])

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