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Part:BBa_K771004

Designed by: Guo Huaqing;Suo Yang   Group: iGEM12_SJTU-BioX-Shanghai   (2012-09-25)
Revision as of 11:02, 29 September 2012 by Jucember (Talk | contribs) (→‎Characterization)

Membrane Anchor 4 : ssDsbA-PDZ Domain-LGT-GBD Domain

Membrane anchor 4, which consists of interacting PDZ domain(BBa_K771103), GBD domain(BBa_K771105) and membrane protein Lgt(BBa_K771102). Membrane Anchor 4 is proved to constitutively aggregate with Membrane Anchor 3 and 5(with and without VVD).

Overview: Membrane Scaffold System

12SJTU-MA1-MA6.png

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

12SJTU-CA345.png

Membrane Anchor 3 (BBa_K771003)and Membrane Anchor 4 constitutively aggregate through PDZ domain(BBa_K771103) and PDZ ligand (BBa_K771104).

Membrane Anchor 4 and Membrane Anchor 5 (with and without VVD) constitutively aggregate through GBD domain(BBa_K771105) and GBD ligand (BBa_K771106).

Design

12SJTU MA4.png

Membrane Anchor 4 could constitutively aggregate with Membrane Anchor 3 and Membrane Anchor 5 through interacting domain and ligand.

Characterization

To testify the constitutive aggregation of Membrane Anchor 3, 4 and 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.

12SJTU Anchor GFP.jpg


Membrane Anchor 3 and Membrane Anchor 4

12SJTU splitegfp34.png
File:12SJTU splitegfp34r.jpg
400px

We fused split EGFP 1 and 2 to Membrane Anchor 4 and Membrane Anchor 3 to test whether Membrane Anchor 3 and Membrane Anchor 4 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 3 and Membrane Anchor 4 could constitutively aggregate through PDZ domain and ligand.

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Membrane Anchor 4 and Membrane Anchor 5 without VVD

12SJTU splitegfp45.png
File:12SJTU splitegfp45r.jpg
400px

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 GBD domain 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.

12SJTU-FATTYACID.png


Related Biobrick

<br\>Membrabe Anchor 1([BBa_K771001]) <br\>Membrabe Anchor 2([BBa_K771002]) <br\>Membrabe Anchor 3([BBa_K771003]) <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 NgoMIV site found at 115
    Illegal AgeI site found at 1174
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 345
    Illegal BsaI site found at 795


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Categories
Parameters
n/aMembrane Anchor 4 : ssDsbA-PDZ Domain-LGT-GBD Domain