Part:BBa_K771003

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

Membrane anchor 3, which consists of SH3 ligand(BBa_K771108), PDZ ligand(BBa_K771104) and membrane protein Lgt(BBa_K771102). Membrane Anchor 3 is proved to constitutively aggregate with Membrane Anchor 2 (with and without MS2)([BBa_K771002])and Membrane Anchor 4 ([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_K771007)and 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

We fused split EGFP 1 and 2 to Membrane Anchor 2 without MS2(BBa_K771007) 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 observationThe 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.

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 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

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 904
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 525