Part:BBa_K771008

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 (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).

Design

Membrane Anchor 5 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 (BBa_K771004).

Characterization

To testify the constitutive aggregation of Membrane Anchor 4 and Membrane Anchor 5, 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 4 and Membrane Anchor 5 without VVD

We fused split EGFP 1 and 2 to Membrane Anchor 4 and Membrane Anchor 5 without VVD (BBa_K771008)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.

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

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 883
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 504